Homodimeric bispecific antibody, preparation method therefor and use thereof

ABSTRACT

Provided is a tetravalent homodimeric bispecific antibody molecule simultaneously targeting an immune effector cell antigen CD3 and a tumor-associated antigen, wherein the bispecific antibody molecule contains, in order from N-terminus to C-terminus, a first single chain Fv, a second single chain Fv and a Fc fragment; wherein the first single chain Fv can specifically bind to the tumor-associated antigen, the second single chain Fv can specifically bind to CD3, and the first and the second single chain Fvs are connected by a linker peptide, while the second single chain Fv and the Fc fragment are directly connected or connected by a linker peptide; and the Fc fragment does not have effector functions such as CDC, ADCC and ADCP.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application claims priority to Chinese patent application No. CN201811294887.4 filed on Nov. 1, 2018, the disclosure of which isincorporated herein by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to the field of immunology and, morespecifically, to an anti-CD3 bispecific antibody that mediates T-cellkilling and the use of such an antibody, particularly in the use thereoffor the treatment of cancer.

BACKGROUND

In 1985, the concept of killing tumor cells using T cells was proposed(Stearz U D et al., Nature, 314: 628-631, 1985). It is generallybelieved that the effective activation of T cells requires a dualsignal, in which the first signal comes from the binding of theMHC-antigen complex to the T-cell receptor TCR-CD3 on theantigen-presenting cell and the second signal is a non-antigen-specificcostimulatory signal resulting from the interaction of the T cell with acostimulatory molecule expressed by the antigen-presenting cell. Due tothe down-regulated expression or even the deletion of MHC on the surfaceof most tumor cells, the tumor cells can escape the immune killing.

The bispecific antibodies can be classified according to the actionmechanism into dual signal blocking type and cell-mediated functionaltype. Generally, the cell-mediated functional bispecific antibody refersto the anti-CD3 bispecific antibody that mediates the T-cell killing.The CD3 molecule is expressed on the surface of all mature T cells, andnon-covalently binds to the TCR to form an intact TCR-CD3 complex, whichjointly participates in the immune response to antigen stimulation andis the most used and the most successful trigger molecule on the surfaceof immune effector cells among bispecific antibodies. The bispecificantibody targeting CD3 can bind to the T cell surface CD3 and the tumorcell surface antigen, respectively, thus shortening the distance betweencytotoxic T cells (Tc or CTL) and tumor cells and directly activating Tcells to induce T cells to directly kill cancer cells instead of relyingon the conventional dual activation signal of T cells. However, theagonistic antibody targeting the T cell antigen CD3, for example, thefirst generation of mouse monoclonal antibody OKT3 targeting human CD3applied to the clinical practice (Kung P et al., Science, 206: 347-349,1979), releases a large number of inflammatory factors such asinterleukin-2 (IL-2), TNF-α, IFN-γ, and interleukin-6 (IL-6) due to thehyperactivation of T cells, which clinically causes a severe “cytokinestorm syndrome” (Hirsch R et al., J. Immunol., 142: 737-743, 1989) andthus resulting in “influenza-like” symptoms characterized by fever,chills, headache, nausea, vomiting, diarrhea, respiratory distress,aseptic meningitis, and hypotension. Thus, how to attenuate or avoid theexcessive cytokine storm is a priority for the development ofbifunctional antibodies targeting CD3.

In recent years, to solve the problem of correctly assembling twodifferent half-antibodies, scientists have designed and developedbispecific antibodies of various structures. Overall, there are twocategories. The one is that the bispecific antibody does not include anFc region. The advantages of bispecific antibodies in such a structureare that they have a small molecular weight, can be expressed inprokaryotic cells, and do not need to be correctly assembled, whiletheir disadvantages are that due to the lack of the antibody Fc fragmentand the relatively low molecular weight, they have a short half-lifeperiod and that such bispecific antibodies are highly susceptible topolymerization, and thus have poor stability and low expression, andthus are limited in the clinical application. Such bispecific antibodiesthat have been reported so far include BiTE, DART, TrandAbs,bi-Nanobody, etc.

The other is that the bispecific antibody retains an Fc domain. Suchbispecific antibodies form an IgG-like structure with a larger molecularstructure and have a longer half-life period due to the FcRn-mediatedendocytosis and recycling process; meanwhile, they also retain some orall of the Fc-mediated effector functions, such as antibody-dependentcell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity(CDC), and antibody-dependent cellular phagocytosis (ADCP). Suchbispecific antibodies that have been reported so far include Triomabs,kih IgG, Cross-mab, orthoFab IgG, DVD IgG, IgG scFv, scFv2-Fc, etc. Asfor anti-CD3 bispecific antibodies, currently, except for configurationsTandAb and scFv-Fv-scFv, other anti-CD3 bispecific antibodies are widelydesigned in the form of univalent anti-CD3, mainly because bivalentanti-CD3 bispecific antibodies can easily lead to hyperactivation andthus induce T cell apoptosis and massive transient release of cytokines(Kuhn C et al, Immunotherapy, 8: 889-906, 2016) and, more seriously,they may also trigger non-antigen-dependent activation of T cells andthus disrupt immune homeostasis. Therefore, most of the anti-CD3bispecific antibodies in the existing art avoid the introduction ofbivalent anti-CD3 antibodies. For example, bispecific antibodies inconfigurations of triFab-Fc, DART-Fc, and BiTE-Fc are designedasymmetrically (that is, heterodimer-type bispecific antibodies) (Z Wuet al, Pharmacology and Therapeutics, 182: 161-175, 2018), but such adesign poses many challenges for downstream production of suchheterodimeric bispecific antibodies, such as the generation of undesiredhomodimers or mismatched impurity molecules, which increase thedifficulty of expression and purification of bispecific antibodies.Although the use of the “knobs-into-holes” technique to some extentsolves the problem of inter-heavy chain mismatching of heterodimericbispecific antibody molecules, “light chain/heavy chain mismatching”brings about another challenge. One strategy for preventing heavychain-light chain mismatching is to interchange the partial domains ofthe light chain and heavy chain of one of Fabs of a bispecific antibodyto form a Crossmab (a hybrid antibody), which can allow selectivepairing between the light chains/heavy chains. However, thedisadvantages of this method are that the generation of mismatchingproducts cannot be completely prevented, and residual fractions of anymismatching molecule are difficult to separate from the products, and inaddition, this method requires a large number of genetic engineeringmodifications such as mutations for two antibody sequences. Thus, thismethod cannot become simple and universal.

Furthermore, for bispecific antibodies including CD3-specific IgG-likestructures, such bispecific antibodies may cause unrestrictedlong-lasting T-cell activation due to their ability to bind to FcγR, andsuch activation is non-target cell-restricted, that is, activatedT-cells can be found in tissues expressing FcγR (e.g., in hematopoietic,lymphoid, and reticuloendothelial systems), whether or not suchbispecific antibodies bind to the target antigen. The systemicactivation of such T cells will be accompanied by a substantial releaseof cytokines, which is a serious adverse effect during the therapeuticapplication of T cells-activated cytokines or antibodies. Therefore,such anti-CD3 bispecific antibodies that mediate the T cell killing needto avoid Fc-mediated systemic activation of T cells, thus allowingimmune effector cells to be restrictedly activated within target celltissues, that is, relying exclusively on the binding of the bispecificantibodies to the corresponding target antigens.

Therefore, there is an urgent need in the art to develop novelbispecific molecules with improved properties in terms of producthalf-life period, stability, safety, and productibility.

SUMMARY

An object of the present disclosure is to provide a tetravalenthomodimer-type bispecific antibody molecule targeting immune effectorcell antigen CD3 and a tumor-associated antigen (TAA). Such a bispecificantibody can significantly inhibit or kill tumor cells in vivo, but hassignificantly reduced non-specific killing effect for normal cells withlow TAA expression, and meanwhile has controlled toxic side effects thatmay be caused by excessive activation of effector cells, andsignificantly improved physicochemical and in vivo stabilities.

In a first aspect of the present disclosure, provided is a bispecificantibody which is a tetravalent homodimer formed by two identicalpolypeptide chains that bind to each other by a covalent bond, whereineach of the two identical polypeptide chains includes, in sequence fromN-terminus to C-terminus, a first single-chain Fv that specificallybinds to a tumor-associated antigen (anti-TAA scFv), a secondsingle-chain Fv that specifically bind to effector cell antigen CD3(anti-CD3 scFv), and an Fc fragment; wherein the first and the secondsingle-chain Fvs are linked by a linker peptide, the second single-chainFv and the Fc fragment are linked directly or by a linker peptide, andthe Fc fragment does not have effector functions.

The first single-chain Fv has specificity to the tumor-associatedantigen and includes a VH domain and a VL domain linked by a linkerpeptide (L1), wherein VH, L1, and VL are arranged in the order ofVH-L1-VL or VL-L1-VH, and the amino acid sequence of the linker peptideL1 is (GGGGX)_(n), wherein X includes Ser or Ala, preferably Ser, and nis a natural number of 1 to 5, preferably 3; Illustratively, thetumor-associated antigen includes, but is not limited to, CD19, CD20,CD22, CD25, CD30, CD33, CD38, CD39, CD40, CD47, CD52, CD73, CD74, CD123,CD133, CD138, BCMA, CA125, CEA, CS1, DLL3, DLL4, EGFR, EpCAM, FLT3,gpA33, GPC-3, Her2, MEGE-A3, NYESO1, PSMA, TAG-72, CIX, folate-bindingprotein, GD2, GD3, GM2, VEGF, VEGFR2, VEGFR3, Cadherin, Integrin,Mesothelin, Claudin18, αVβ3, α5β1, ERBB3, c-MET, IGF1R, EPHA3, TRAILR1,TRAILR2, RANKL, B7 protein family, Mucin, FAP, and Tenascin; preferably,the tumor-associated antigen is CD19, CD20, CD22, CD30, CD38, BCMA, CS1,EpCAM, CEA, Her2, EGFR, CA125, Mucin1, GPC-3, and Mesothelin.

For example, some preferred amino acid sequences of the VH domain andits complementary determining regions (HCDR1, HCDR2, and HCDR3) andamino acid sequences of the VL domain and its complementary determiningregions (LCDR1, LCDR2 and LCDR3) of a first single-chain Fv targetingthe tumor-associated antigen are exemplified in Table 6-1 of the presentdisclosure.

Preferably, the first single-chain Fv specifically binds to CD19 and isselected from the group consisting of:

-   -   (i) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 9, 10, and 11, respectively or having sequences that        are substantially identical to (for example, are at least 80%,        85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one        or more amino acid substitutions (for example, conservative        substitutions) than) any of SEQ ID NOs: 9, 10, and 11; and a VL        domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID        NOs: 12, 13, and 14, respectively or having sequences that are        substantially identical to (for example, are at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or        more amino acid substitutions (for example, conservative        substitutions) than) any of SEQ ID NOs: 12, 13, and 14;    -   (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 17, 18, and 19, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 17, 18, and        19; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown        in SEQ ID NOs: 20, 21, and 22, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 20, 21, and        22;    -   (iii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 25, 26, and 27, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 25, 26, and        27; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown        in SEQ ID NOs: 28, 29, and 30, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 28, 29, and        30; and    -   (iv) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 33, 34, and 35, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 33, 34, and        35; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown        in SEQ ID NOs: 36, 37, and 38, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 36, 37, and        38.

Preferably, the first single-chain Fv specifically binds to CD20 and isselected from the group consisting of:

-   -   (i) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 41, 42, and 43, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 41, 42, and        43; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown        in SEQ ID NOs: 44, 45, and 46, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 44, 45, and        46;    -   (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 49, 50, and 51, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 49, 50, and        51; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown        in SEQ ID NOs: 52, 53, and 54, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 52, 53, and        54;    -   (iii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 57, 58, and 59, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 57, 58, and        59; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown        in SEQ ID NOs: 60, 61, and 62, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 60, 61, and        62; and    -   (iv) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 65, 66, and 67, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 65, 66, and        67; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown        in SEQ ID NOs: 68, 69, and 70, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 68, 69, and        70.

Preferably, the first single-chain Fv specifically binds to CD22 and isselected from the group consisting of:

-   -   (i) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 73, 74, and 75, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 73, 74, and        75; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown        in SEQ ID NOs: 76, 77, and 78, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 76, 77, and        78; and    -   (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 81, 82, and 83, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 81, 82, and        83; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown        in SEQ ID NOs: 84, 85, and 86, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 84, 85, and        86.

Preferably, the first single-chain Fv specifically binds to CD30 and isselected from the group consisting of:

-   -   (i) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 89, 90, and 91, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 89, 90, and        91; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown        in SEQ ID NOs: 92, 93, and 94, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 92, 93, and        94; and    -   (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 97, 98, and 99, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 97, 98, and        99; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown        in SEQ ID NOs: 100, 101, and 102, respectively or having        sequences that are substantially identical to (for example, are        at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar        to or have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 100, 101,        and 102.

Preferably, the first single-chain Fv specifically binds to EpCAM and isselected from the group consisting of:

-   -   (i) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 105, 106, and 107, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 105, 106,        and 107; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as        shown in SEQ ID NOs: 108, 109, and 110, respectively or having        sequences that are substantially identical to (for example, are        at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar        to or have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 108, 109,        and 110; and    -   (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 113, 114, and 115, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 113, 114,        and 115; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as        shown in SEQ ID NOs: 116, 117, and 118, respectively or having        sequences that are substantially identical to (for example, are        at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar        to or have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 116, 117,        and 118.

Preferably, the first single-chain Fv specifically binds to CEA and isselected from the group consisting of:

-   -   (i) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 121, 122, and 123, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 121, 122,        and 123; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as        shown in SEQ ID NOs: 124, 125, and 126, respectively or having        sequences that are substantially identical to (for example, are        at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar        to or have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 124, 125,        and 126;    -   (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 129, 130, and 131, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 129, 130,        and 131; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as        shown in SEQ ID NOs: 132, 133, and 134, respectively or having        sequences that are substantially identical to (for example, are        at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar        to or have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 132, 133,        and 134; and    -   (iii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 137, 138, and 139, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 137, 138,        and 139; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as        shown in SEQ ID NOs: 140, 141, and 142, respectively or having        sequences that are substantially identical to (for example, are        at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar        to or have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 140, 141,        and 142.

Preferably, the first single-chain Fv specifically binds to Her2 and isselected from the group consisting of:

-   -   (i) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 145, 146, and 147, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 145, 146,        and 147; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as        shown in SEQ ID NOs: 148, 149, and 150, respectively or having        sequences that are substantially identical to (for example, are        at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar        to or have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 148, 149,        and 150;    -   (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 153, 154, and 155, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 153, 154,        and 155; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as        shown in SEQ ID NOs: 156, 157, and 158, respectively or having        sequences that are substantially identical to (for example, are        at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar        to or have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 156, 157,        and 158; and    -   (iii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 161, 162, and 163, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 161, 162,        and 163; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as        shown in SEQ ID NOs: 164, 165, and 166, respectively or having        sequences that are substantially identical to (for example, are        at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar        to or have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 164, 165,        and 166.

Preferably, the first single-chain Fv specifically binds to EGFR and isselected from the group consisting of:

-   -   (i) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 169, 170, and 171, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 169, 170,        and 171; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as        shown in SEQ ID NOs: 172, 173, and 174, respectively or having        sequences that are substantially identical to (for example, are        at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar        to or have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 172, 173,        and 174;    -   (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 177, 178, and 179, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 177, 178,        and 179; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as        shown in SEQ ID NOs: 180, 181, and 182, respectively or having        sequences that are substantially identical to (for example, are        at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar        to or have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 180, 181,        and 182; and    -   (iii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 185, 186, and 187, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 185, 186,        and 187; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as        shown in SEQ ID NOs: 188, 189, and 190, respectively or having        sequences that are substantially identical to (for example, are        at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar        to or have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 188, 189,        and 190.

Preferably, the first single-chain Fv specifically binds to GPC-3; theVH domain of the first single-chain Fv includes HCDR1, HCDR2, and HCDR3as shown in SEQ ID NOs: 193, 194, and 195, respectively or havingsequences that are substantially identical to (for example, are at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one ormore amino acid substitutions (for example, conservative substitutions)than) any of SEQ ID NOs: 193, 194, and 195; and the VL domain of thefirst single-chain Fv includes LCDR1, LCDR2, and LCDR3 as shown in SEQID NOs: 196, 197, and 198, respectively or having sequences that aresubstantially identical to (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) anyof SEQ ID NOs: 196, 197, and 198.

Preferably, the first single-chain Fv specifically binds to Mesothelin;the VH domain of the first single-chain Fv includes HCDR1, HCDR2, andHCDR3 as shown in SEQ ID NOs: 201, 202, and 203, respectively or havingsequences that are substantially identical to (for example, are at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one ormore amino acid substitutions (for example, conservative substitutions)than) any of SEQ ID NOs: 201, 202, and 203; and the VL domain of thefirst single-chain Fv includes LCDR1, LCDR2, and LCDR3 as shown in SEQID NOs: 204, 205, and 206, respectively or having sequences that aresubstantially identical to (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) anyof SEQ ID NOs: 204, 205, and 206.

Preferably, the first single-chain Fv specifically binds to Mucin1 andis selected from the group consisting of:

-   -   (i) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 209, 210, and 211, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 209, 210,        and 211; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as        shown in SEQ ID NOs: 212, 213, and 214, respectively or having        sequences that are substantially identical to (for example, are        at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar        to or have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 212, 213,        and 214; and    -   (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in        SEQ ID NOs: 217, 218, and 219, respectively or having sequences        that are substantially identical to (for example, are at least        80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or        have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 217, 218,        and 219; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as        shown in SEQ ID NOs: 220, 221, and 222, respectively or having        sequences that are substantially identical to (for example, are        at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar        to or have one or more amino acid substitutions (for example,        conservative substitutions) than) any of SEQ ID NOs: 220, 221,        and 222.

Preferably, the first single-chain Fv specifically binds to CA125; theVH domain of the first single-chain Fv includes HCDR1, HCDR2, and HCDR3as shown in SEQ ID NOs: 225, 226, and 227, respectively or havingsequences that are substantially identical to (for example, are at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one ormore amino acid substitutions (for example, conservative substitutions)than) any of SEQ ID NOs: 225, 226, and 227; and the VL domain of thefirst single-chain Fv includes LCDR1, LCDR2, and LCDR3 as shown in SEQID NOs: 228, 229, and 230, respectively or having sequences that aresubstantially identical the (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) toany of SEQ ID NOs: 228, 229, and 230.

Preferably, the first single-chain Fv specifically binds to BCMA; the VHdomain of the first single-chain Fv includes HCDR1, HCDR2, and HCDR3 asshown in SEQ ID NOs: 233, 234, and 235, respectively or having sequencesthat are substantially identical to (for example, are at least 80%, 85%,90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or moreamino acid substitutions (for example, conservative substitutions) than)any of SEQ ID NOs: 233, 234, and 235; and the VL domain of the firstsingle-chain Fv includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs:236, 237, and 238, respectively or having sequences that aresubstantially identical to (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) anyof SEQ ID NOs: 236, 237, and 238.

More preferably, the first single-chain Fv specifically binds to CD19and is selected from the group consisting of:

-   -   (i) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 15 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 15; and a VL domain comprising an amino acid sequence        as shown in SEQ ID NO: 16 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 16;    -   (ii) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 23 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 23; and a VL domain comprising an amino acid sequence        as shown in SEQ ID NO: 24 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 24;    -   (iii) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 31 or having a sequence that is substantially        identical (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than) to        SEQ ID NO: 31; and a VL domain comprising an amino acid sequence        as shown in SEQ ID NO: 32 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 32; and    -   (iv) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 39 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 39; and a VL domain comprising an amino acid sequence        as shown in SEQ ID NO: 40 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 40.

More preferably, the first single-chain Fv specifically binds to CD20and is selected from the group consisting of:

-   -   (i) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 47 or having a sequence that is substantially        identical (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than) to        SEQ ID NO: 47; and a VL domain comprising an amino acid sequence        as shown in SEQ ID NO: 48 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 48;    -   (ii) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 55 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 55; and a VL domain comprising an amino acid sequence        as shown in SEQ ID NO: 56 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 56;    -   (iii) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 63 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 63; and a VL domain comprising an amino acid sequence        as shown in SEQ ID NO: 64 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 64; and    -   (iv) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 71 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 71; and a VL domain comprising an amino acid sequence        as shown in SEQ ID NO: 72 or a sequence substantially identical        to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%,        99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 72.

More preferably, the first single-chain Fv specifically binds to CD22and is selected from the group consisting of:

-   -   (i) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 79 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 79; and a VL domain comprising an amino acid sequence        as shown in SEQ ID NO: 80 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 80; and    -   (ii) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 87 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 87; and a VL domain comprising an amino acid sequence        as shown in SEQ ID NO: 88 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 88.

More preferably, the first single-chain Fv specifically binds to CD30and is selected from the group consisting of:

-   -   (i) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 95 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 95; anda VL domain comprising an amino acid sequence        as shown in SEQ ID NO: 96 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 96; and    -   (ii) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 103 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 103; and a VL domain comprising an amino acid        sequence as shown in SEQ ID NO: 104 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 104.

More preferably, the first single-chain Fv specifically binds to EpCAMand is selected from the group consisting of:

-   -   (i) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 111 or having a sequence that is substantially        identical (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or as one or more amino acid        substitutions (for example, conservative substitutions) than) to        SEQ ID NO: 111; and a VL domain comprising an amino acid        sequence as shown in SEQ ID NO: 112 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 112; and    -   (ii) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 119 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 119; and a VL domain comprising an amino acid        sequence as shown in SEQ ID NO: 120 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 120.

More preferably, the first single-chain Fv specifically binds to CEA andis selected from the group consisting of:

-   -   (i) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 127 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 127; and a VL domain comprising an amino acid        sequence as shown in SEQ ID NO: 128 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 128;    -   (ii) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 135 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 135; and a VL domain comprising an amino acid        sequence as shown in SEQ ID NO: 136 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 136; and    -   (iii) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 143 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 143; and a VL domain comprising an amino acid        sequence as shown in SEQ ID NO: 144 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 144.

More preferably, the first single-chain Fv specifically binds to Her2and is selected from the group consisting of:

-   -   (i) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 151 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 151; and a VL domain comprising an amino acid        sequence as shown in SEQ ID NO: 152 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 152;    -   (ii) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 159 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 159; and a VL domain comprising an amino acid        sequence as shown in SEQ ID NO: 160 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 160; and    -   (iii) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 167 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 167; and a VL domain comprising an amino acid        sequence as shown in SEQ ID NO: 168 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 168.

More preferably, the first single-chain Fv specifically binds to EGFRand is selected from the group consisting of:

-   -   (i) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 175 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 175; and a VL domain comprising an amino acid        sequence as shown in SEQ ID NO: 176 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 176;    -   (ii) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 183 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 183; and a VL domain comprising an amino acid        sequence as shown in SEQ ID NO: 184 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 184; and    -   (iii) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 191 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 191; and a VL domain comprising an amino acid        sequence as shown in SEQ ID NO: 192 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 192.

More preferably, the first single-chain Fv specifically binds to GPC-3;the VH domain of the first single-chain Fv includes an amino acidsequence as shown in SEQ ID NO: 199 or having a sequence that issubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:199; and the VL domain of the first single-chain Fv includes an aminoacid sequence as shown in SEQ ID NO: 200 or having a sequence that issubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:200.

More preferably, the first single-chain Fv specifically binds toMesothelin; the VH domain of the first single-chain Fv includes an aminoacid sequence as shown in SEQ ID NO: 207 or having a sequence that issubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:207; and the VL domain of the first single-chain Fv includes an aminoacid sequence as shown in SEQ ID NO: 208 or having a sequence that issubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:208.

More preferably, the first single-chain Fv specifically binds to Mucin1and is selected from the group consisting of:

-   -   (i) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 215 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 215; and a VL domain comprising an amino acid        sequence as shown in SEQ ID NO: 216 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 216; and    -   (ii) a VH domain comprising an amino acid sequence as shown in        SEQ ID NO: 223 or having a sequence that is substantially        identical to (for example, is at least 80%, 85%, 90%, 92%, 95%,        97%, 98%, 99% or more similar to or has one or more amino acid        substitutions (for example, conservative substitutions) than)        SEQ ID NO: 223; and a VL domain comprising an amino acid        sequence as shown in SEQ ID NO: 224 or having a sequence that is        substantially identical to (for example, is at least 80%, 85%,        90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or        more amino acid substitutions (for example, conservative        substitutions) than) SEQ ID NO: 224.

More preferably, the first single-chain Fv specifically binds to CA125;the VH domain of the first single-chain Fv includes an amino acidsequence as shown in SEQ ID NO: 231 or having a sequence that issubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:231; and the VL domain of the first single-chain Fv includes an aminoacid sequence as shown in SEQ ID NO: 232 or having a sequence that issubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:232.

More preferably, the first single-chain Fv specifically binds to BCMA;the VH domain of the first single-chain Fv includes an amino acidsequence as shown in SEQ ID NO: 239 or having a sequence that issubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:239; and the VL domain of the first single-chain Fv includes an aminoacid sequence as shown in SEQ ID NO: 240 or having a sequence that issubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:240.

The linker peptide (L2) connecting the first single-chain Fv and thesecond single-chain Fv in the present disclosure consists of a flexiblepeptide and a rigid peptide.

Furthermore, the flexible peptide includes two or more amino acids thatare preferably selected from the group consisting of the following aminoacids: Gly(G), Ser(S), Ala(A), and Thr(T). More preferably, the flexiblepeptide includes G and S residues. Most preferably, the amino acidcomposition structure of the flexible peptide has a general formulaG_(x)S_(y)(GGGGS)_(z), where x, y, and z are integers greater than orequal to 0, and x+y+z >1. For example, in a preferred embodiment, theamino acid sequence of the flexible peptide is G₂(GGGGS)₃.

Furthermore, the rigid peptide is derived from a full-length sequence(as shown in SEQ ID NO: 257) consisting of amino acids at positions 118to 145 of the carboxy-terminus of the natural human chorionicgonadotropin beta-subunit, or a truncated fragment thereof (hereinaftercollectively referred to as CTP). Preferably, the CTP rigid peptideincludes 10 amino acids at the N-terminal of SEQ ID NO: 257, that is,SSSSKAPPPS (CTP¹); or the CTP rigid peptide includes 14 amino acids atthe C-terminal of SEQ ID NO: 257, that is, SRLPGPSDTPILPQ (CTP²); asanother example, in another embodiment, the CTP rigid peptide includes16 amino acids at the N-terminal of SEQ ID NO: 257, that is,SSSSKAPPPSLPSPSR (CTP³); for another example, in another embodiment, theCTP rigid peptide includes 28 amino acids that begin at the position 118and end at position 145 of the human chorionic gonadotropinbeta-subunit, that is, SSSSKAPPPSLPSPSRLPGPSDTPILPQ (CTP⁴).

For example, some preferred amino acid sequences of the linker peptideL2 that links the first single-chain Fv and second single-chain Fv areexemplified listed in Table 6-3 of the present disclosure.

In a preferred embodiment of the present disclosure, the linker peptidehas an amino acid sequence as shown in SEQ ID NO: 258, wherein the aminoacid composition of the flexible peptide is G₂(GGGGS)₃, and the aminoacid composition of the rigid peptide is SSSSKAPPPS (that is, CTP¹).

The second single-chain Fv has specificity to immune effector cellantigen CD3 and includes a VH domain and a VL domain linked by a linkerpeptide (L3), wherein VH, L3, and VL are arranged in the order ofVH-L3-VL or VL-L3-VH, and the amino acid sequence of the linker peptideL3 is (GGGGX)_(n), wherein X includes Ser or Ala, preferably Ser; and nis a natural number of 1 to 5, preferably 3;

Preferably, the second single-chain Fv of the bispecific antibody bindsto an effector cell at an EC₅₀ value greater than about 50 nM, orgreater than 100 nM, or greater than 300 nM, or greater than 500 nM inan in vitro FACS binding affinity assay; more preferably, the secondsingle-chain Fv of the bispecific antibody not only binds to human CD3,but also specifically binds to CD3 of a cynomolgus monkey or a rhesusmonkey. In a preferred embodiment of the present disclosure, thebispecific antibody specifically binds to the effector cell at an EC₅₀value of 132.3 nM.

For example, some preferred amino acid sequences of the VH domain andits complementary determining regions (HCDR1, HCDR2, and HCDR3) andamino acid sequences of the VL domain and its complementary determiningregions (LCDR1, LCDR2 and LCDR3) of the anti-CD3 scFv are exemplified inTable 6-2 of the present disclosure.

Preferably, the second single-chain Fv specifically binds to CD3; the VHdomain of the second single-chain Fv includes HCDR1, HCDR2, and HCDR3 asshown in SEQ ID NOs: 241, 242, and 243, respectively or having sequencesthat are substantially identical to (for example, are at least 80%, 85%,90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or moreamino acid substitutions (for example, conservative substitutions) than)SEQ ID NOs: 241, 242, and 243; and the VL domain of the secondsingle-chain Fv includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs:244, 245, and 246, respectively or having sequences that aresubstantially identical to (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) SEQID NOs: 244, 245, and 246.

Preferably, the second single-chain Fv specifically binds to CD3; the VHdomain of the second single-chain Fv includes HCDR1, HCDR2, and HCDR3 asshown in SEQ ID NOs: 249, 250, and 251, respectively or having sequencesare substantially identical to (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) SEQID NOs: 249, 250, and 251; and the VL domain of the second single-chainFv includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 252, 253,and 254, respectively or having sequences that are substantiallyidentical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or more similar to or have one or more amino acid substitutions(for example, conservative substitutions) than) SEQ ID NOs: 252, 253,and 254.

More preferably, the second single-chain Fv specifically binds to CD3;the VH domain of the second single-chain Fv includes an amino acidsequence as shown in SEQ ID NO: 247 or having a sequence that issubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:247; and the VL domain of the second single-chain Fv includes an aminoacid sequence as shown in SEQ ID NO: 248 or having a sequence that issubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:248.

More preferably, the second single-chain Fv specifically binds to CD3;the VH domain of the second single-chain Fv includes an amino acidsequence as shown in SEQ ID NO: 255 or having a sequence that issubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:255; and the VL domain of the second single-chain Fv includes an aminoacid sequence as shown in SEQ ID NO: 256 or having a sequence that issubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:256.

The Fc fragment of the present disclosure is linked to the secondsingle-chain Fv directly or by the linker peptide L4, and the linkerpeptide L4 includes 1 to 20 amino acids, and preferably selected fromthe following amino acids: Gly(G), Ser(S), Ala(A), and Thr(T); morepreferably, the linker peptide L4 is selected from Gly(G) and Ser(S);further preferably, the linker peptide L4 consists of (GGGGS)_(n),wherein n=1, 2, 3 or 4. In a preferred embodiment of the presentdisclosure, the Fc fragment is directly linked to the secondsingle-chain Fv.

In another aspect, the Fc fragment of the present disclosure includes ahinge region, a CH2 domain, and a CH3 domain from a human immunoglobulinheavy chain constant region. For example, in some embodiments, the Fcfragment of the present disclosure is selected from heavy chain constantregions of human IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE;particularly selected from heavy chain constant regions of human IgG1,IgG2, IgG3, and IgG4, and more particularly selected from a heavy chainconstant region of human IgG1 or IgG4; and the Fc fragment has one ormore amino acid substitutions, deletions or additions (for example, atmost 20, at most 15, at most 10, or at most 5 substitutions, deletionsor additions) than a natural sequence from which the Fc fragment isderived.

In some preferred embodiments, the Fc fragment is changed, for example,mutated to modify the properties of the bispecific antibody molecule ofthe present disclosure (for example, to change one or more of thefollowing properties: Fc receptor binding, antibody glycosylation, aneffector cell function or a complement function).

For example, the bispecific antibody provided by the present disclosureincludes an Fc variant containing amino acid substitutions, deletions oradditions that change (for example, reduce or eliminate) effectorfunctions. Fc region of the antibody mediates several important effectorfunctions, such as ADCC, ADCP, and CDC. Methods for changing theaffinity of the antibody to an effector ligand (such as FcγR or acomplement C1q) by substituting amino acid residues in the Fc region ofthe antibody to change the effector functions are known in the art (see,for example, EP 388151A1; U.S. Pat. Nos. 5,648,260; 5,624,821; Natsume Aet al., Cancer Res., 68: 3863-3872, 2008; Idusogie E E et al., J.Immunol., 166: 2571-2575, 2001; Lazar G A et al., PNAS, 103: 4005-4010,2006; Shields R L et al., JBC, 276: 6591-6604, 2001; Stavenhagen J B etal., Cancer Res., 67: 8882-8890, 2007; Stavenhagen J B et al., Advan.Enzyme. Regul., 48: 152-164, 2008; Alegre M L et al., J. Immunol., 148:3461-3468, 1992; Kaneko E et al., Biodrugs, 25: 1-11, 2011). In somepreferred embodiments of the present disclosure, amino acid L235 (EUnumbering) in the constant region of the antibody is modified to changean interaction with an Fc receptor, such as L235E or L235A. In someother preferred embodiments, amino acids 234 and 235 in the constantregion of the antibody are modified simultaneously, such as L234A andL235A (L234A/L235A) (EU numbering).

For example, the bispecific antibody provided by the present disclosuremay include an Fc variant containing amino acid substitutions, deletionsor additions that extend a circulating half-life. Studies show thatM252Y/S254T/T256E, M428L/N434S or T250Q/M428L can extend the half-lifeof the antibody in primates. For more mutation sites included in the Fcvariant with enhanced binding affinity to a neonatal receptor (FcRn),see Chinese invention patent CN 201280066663.2, U.S. 2005/0014934A1, WO97/43316, U.S. Pat. Nos. 5,869,046, 5,747,030 and WO 96/32478. In somepreferred embodiments of the present disclosure, amino acid M428 (EUnumbering) in the constant region of the antibody is modified to enhancethe binding affinity for the FcRn receptor, such as M428L. In some otherpreferred embodiments, amino acids 250 and 428 (EU numbering) in theconstant region of the antibody are modified simultaneously, such asT250Q and M428L (T250Q/M428L).

For example, the bispecific antibody provided by the present disclosuremay also include an Fc variant containing amino acid substitutions,deletions or additions that may reduce or eliminate Fc glycosylation.For example, the Fc variant contains reduced glycosylation of theN-linked glycan normally present at amino acid site 297 (EU numbering).The glycosylation at position N297 has a great effect on the activity ofIgG. If the glycosylation at this position is eliminated, theconformation of the upper half of CH2 of an IgG molecule is affected,thus losing the ability of binding to FcγRs and affecting the biologicalactivity related to the antibody. In some preferred embodiments of thepresent disclosure, amino acid N297 (EU numbering) in the constantregion of human IgG is modified to avoid the glycosylation of theantibody, such as N297A.

For example, the bispecific antibody provided by the present disclosuremay also include an Fc variant containing amino acid substitutions,deletions or additions that eliminate charge heterogeneity. Variouspost-translational modifications during expression in engineered cellswill cause the charge heterogeneity of monoclonal antibodies. Theheterogeneity of lysine at C-terminus of an IgG antibody is one of themain reasons for charge heterogeneity. Lysine K at C-terminus of a heavychain may be deleted at a certain proportion during the production ofthe antibody, resulting in charge heterogeneity and affecting thestability, effectiveness, immunogenicity or pharmacokinetic of theantibody. In some preferred embodiments of the present disclosure, K447(EU numbering) at the C-terminus of the IgG antibody is removed ordeleted to eliminate the charge heterogeneity of the antibody andimprove the homogeneity of the expressed product.

The amino acid sequences of some preferred Fc fragments are exemplarilylisted in Table 6-4 of the present disclosure. Compared with abispecific antibody containing the Fc region of wild-type human IgG, thebispecific antibody provided by the present disclosure contains an Fcfragment that exhibits reduced affinity for at least one of human FcγRs(FcγRI, FcγRIIa, or FcγRIIIa) and C1q, and has reduced effector cellfunctions or complement functions. For example, in a preferredembodiment of the present disclosure, the bispecific antibody includesan Fc fragment that is derived from human IgG1, has L234A and L235Asubstitutions (L234A/L235A), and exhibits reduced binding ability forFcγRI. In addition, the Fc fragment included in the bispecific antibodyprovided by the present disclosure may also contain amino acidsubstitutions that change one or more other properties (e.g., ability ofbinding to the FcRn receptor, the glycosylation of the antibody or thecharge heterogeneity of the antibody). For example, in a preferredembodiment of the present disclosure, the Fc fragment has an amino acidsequence as shown in SEQ ID NO: 263, which has amino acid substitutionsL234A/L235A/T250Q/N297A/P331S/M428L and a deleted or removed K447compared with the native sequence from which it is derived.

The bispecific antibody molecule of the present disclosure is atetravalent homodimer formed by two identical polypeptide chains thatbind to each other by an interchain disulfide bond in the hing region ofthe Fc fragment, wherein each polypeptide chain consists of, in sequencefrom N-terminus to C-terminus, an anti-TAA scFv, a linker peptide, ananti-CD3 scFv, and an Fc fragment.

For example, the amino acid sequences of some preferred bispecificantibodies are exemplified in Table 6-5 of the present disclosure.

In a preferred embodiment of the present disclosure, the bispecificantibody binds to human CD19 and CD3 and has an amino acid sequence asfollows:

-   -   (i) a sequence as shown in SEQ ID NO: 264;    -   (ii) a sequence with one or more substitutions, deletions or        additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or        additions) compared to the sequence as shown in SEQ ID NO: 264;        or    -   (iii) a sequence with at least 80%, at least 85%, at least 90%,        at least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99% or        100% sequence identity relative to the sequence as shown in SEQ        ID NO: 264;

In some preferred embodiments, the substitutions in (ii) areconservative substitutions.

In a preferred embodiment of the present disclosure, the bispecificantibody binds to human CD19 and CD3 and has an amino acid sequence asfollows:

-   -   (i) a sequence as shown in SEQ ID NO: 283;    -   (ii) a sequence with one or more substitutions, deletions or        additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or        additions) compared to the sequence as shown in SEQ ID NO: 283;        or    -   (iii) a sequence with at least 80%, at least 85%, at least 90%,        at least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99% or        100% sequence identity to the sequence as shown in SEQ ID NO:        283.

In some preferred embodiments, the substitutions in (ii) areconservative substitutions.

In a preferred embodiment of the present disclosure, the bispecificantibody binds to human CD20 and CD3 and has an amino acid sequence asfollows:

-   -   (i) a sequence as shown in SEQ ID NO: 266;    -   (ii) a sequence with one or more substitutions, deletions or        additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or        additions) compared to the sequence as shown in SEQ ID NO: 266;        or    -   (iii) a sequence with at least 80%, at least 85%, at least 90%,        at least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99% or        100% sequence identity to the sequence as shown in SEQ ID NO:        266.

In some preferred embodiments, the substitutions in (ii) areconservative substitutions.

In a preferred embodiment of the present disclosure, the bispecificantibody binds to human CD22 and CD3 and has an amino acid sequence asfollows:

-   -   (i) a sequence as shown in SEQ ID NO: 268;    -   (ii) a sequence with one or more substitutions, deletions or        additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or        additions) compared to the sequence as shown in SEQ ID NO: 268;        or    -   (iii) a sequence with at least 80%, at least 85%, at least 90%,        at least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99% or        100% sequence identity to the sequence as shown in SEQ ID NO:        268.

In some preferred embodiments, the substitutions in (ii) areconservative substitutions.

In a preferred embodiment of the present disclosure, the bispecificantibody binds to human CD30 and CD3 and has an amino acid sequence asfollows:

-   -   (i) a sequence as shown in SEQ ID NO: 270;    -   (ii) a sequence with one or more substitutions, deletions or        additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or        additions) compared to the sequence as shown in SEQ ID NO: 270;        or    -   (iii) a sequence with at least 80%, at least 85%, at least 90%,        at least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99% or        100% sequence identity to the sequence as shown in SEQ ID NO:        270.

In some preferred embodiments, the substitutions in (ii) areconservative substitutions.

In a preferred embodiment of the present disclosure, the bispecificantibody binds to human EpCAM and CD3 and has an amino acid sequence asfollows:

-   -   (i) a sequence as shown in SEQ ID NO: 272;    -   (ii) a sequence with one or more substitutions, deletions or        additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or        additions) compared to the sequence as shown in SEQ ID NO: 272;        or    -   (iii) a sequence with at least 80%, at least 85%, at least 90%,        at least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99% or        100% sequence identity to the sequence as shown in SEQ ID NO:        272.

In some preferred embodiments, the substitutions in (ii) areconservative substitutions.

In a preferred embodiment of the present disclosure, the bispecificantibody binds to human CEA and CD3 and has an amino acid sequence asfollows:

-   -   (i) a sequence as shown in SEQ ID NO: 274;    -   (ii) a sequence with one or more substitutions, deletions or        additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or        additions) compared to the sequence as shown in SEQ ID NO: 274;        or    -   (iii) a sequence with at least 80%, at least 85%, at least 90%,        at least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99% or        100% sequence identity to the sequence as shown in SEQ ID NO:        274.

In some preferred embodiments, the substitutions in (ii) areconservative substitutions.

In a preferred embodiment of the present disclosure, the bispecificantibody binds to human Her2 and CD3 and has an amino acid sequence asfollows:

-   -   (i) a sequence as shown in SEQ ID NO: 8;    -   (ii) a sequence with one or more substitutions, deletions or        additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or        additions) compared to the sequence as shown in SEQ ID NO: 8; or    -   (iii) a sequence with at least 80%, at least 85%, at least 90%,        at least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99% or        100% sequence identity to the sequence as shown in SEQ ID NO: 8.

In some preferred embodiments, the substitutions in (ii) areconservative substitutions.

In a preferred embodiment of the present disclosure, the bispecificantibody binds to human EGFR and CD3 and has an amino acid sequence asfollows:

-   -   (i) a sequence as shown in SEQ ID NO: 277;    -   (ii) a sequence with one or more substitutions, deletions or        additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or        additions) compared to the sequence as shown in SEQ ID NO: 277;        or    -   (iii) a sequence with at least 80%, at least 85%, at least 90%,        at least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99% or        100% sequence identity to the sequence as shown in SEQ ID NO:        277.

In some preferred embodiments, the substitutions in (ii) areconservative substitutions.

In a preferred embodiment of the present disclosure, the bispecificantibody binds to human GPC-3 and CD3 and has an amino acid sequence asfollows:

-   -   (i) a sequence as shown in SEQ ID NO: 279;    -   (ii) a sequence with one or more substitutions, deletions or        additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or        additions) compared to the sequence as shown in SEQ ID NO: 279;        or    -   (iii) a sequence with at least 80%, at least 85%, at least 90%,        at least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99% or        100% sequence identity to the sequence as shown in SEQ ID NO:        279.

In some preferred embodiments, the substitutions in (ii) areconservative substitutions.

In a preferred embodiment of the present disclosure, the bispecificantibody binds to human Mesothelin and CD3 and has an amino acidsequence as follows:

-   -   (i) a sequence as shown in SEQ ID NO: 281;    -   (ii) a sequence with one or more substitutions, deletions or        additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or        additions) compared to the sequence as shown in SEQ ID NO: 281;        or    -   (iii) a sequence with at least 80%, at least 85%, at least 90%,        at least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99% or        100% sequence identity to the sequence as shown in SEQ ID NO:        281.

In some preferred embodiments, the substitutions in (ii) areconservative substitutions.

In a preferred embodiment of the present disclosure, the bispecificantibody binds to human Mucin1 and CD3 and has an amino acid sequence asfollows:

-   -   (i) a sequence as shown in SEQ ID NO: 285;    -   (ii) a sequence with one or more substitutions, deletions or        additions (such as 1, 2, 3, 4 or 5 substitutions, deletions or        additions) compared to the sequence as shown in SEQ ID NO: 285;        or    -   (iii) a sequence with at least 80%, at least 85%, at least 90%,        at least 91%, at least 92%, at least 93%, at least 94%, at least        95%, at least 96%, at least 97%, at least 98%, at least 99% or        100% sequence identity to the sequence as shown in SEQ ID NO:        285.

In some preferred embodiments, the substitutions in (ii) areconservative substitutions.

In a second aspect of the present disclosure, a DNA molecule encodingthe bispecific antibody as described above is provided.

In a preferred embodiment of the present disclosure, the DNA moleculeencoding the bispecific antibody as described above is a nucleotidesequence as shown in SEQ ID NO: 265.

In a preferred embodiment of the present disclosure, the DNA moleculeencoding the bispecific antibody as described above is a nucleotidesequence as shown in SEQ ID NO: 267.

In a preferred embodiment of the present disclosure, the DNA moleculeencoding the bispecific antibody as described above is a nucleotidesequence as shown in SEQ ID NO: 269.

In a preferred embodiment of the present disclosure, the DNA moleculeencoding the bispecific antibody as described above is a nucleotidesequence as shown in SEQ ID NO: 271.

In a preferred embodiment of the present disclosure, the DNA moleculeencoding the bispecific antibody as described above is a nucleotidesequence as shown in SEQ ID NO: 273.

In a preferred embodiment of the present disclosure, the DNA moleculeencoding the bispecific antibody as described above is a nucleotidesequence as shown in SEQ ID NO: 275.

In a preferred embodiment of the present disclosure, the DNA moleculeencoding the bispecific antibody as described above is a nucleotidesequence as shown in SEQ ID NO: 276.

In a preferred embodiment of the present disclosure, the DNA moleculeencoding the bispecific antibody as described above is a nucleotidesequence as shown in SEQ ID NO: 278.

In a preferred embodiment of the present disclosure, the DNA moleculeencoding the bispecific antibody as described above is a nucleotidesequence as shown in SEQ ID NO: 280.

In a preferred embodiment of the present disclosure, the DNA moleculeencoding the bispecific antibody as described above is a nucleotidesequence as shown in SEQ ID NO: 282.

In a preferred embodiment of the present disclosure, the DNA moleculeencoding the bispecific antibody as described above is a nucleotidesequence as shown in SEQ ID NO: 284.

In a preferred embodiment of the present disclosure, the DNA moleculeencoding the bispecific antibody as described above is a nucleotidesequence as shown in SEQ ID NO: 286.

In a third aspect of the present disclosure, a vector comprising the DNAmolecule as described above is provided.

In a fourth aspect of the present disclosure, a host cell comprising thevector as described above is provided; the host cell includes aprokaryotic cell, a yeast or a mammalian cell, such as a CHO cell, anNS0 cell or another mammalian cell, preferably a CHO cell.

In a fifth aspect of the present disclosure, provided is apharmaceutical composition, comprising the bispecific antibody asdescribed above and a pharmaceutically acceptable excipient, carrier ordiluent.

In a sixth aspect of the present disclosure, further provided a methodfor preparing the bispecific antibody as described in the presentdisclosure, comprising: (a) obtaining a fusion gene of the bispecificantibody to construct an expression vector of the bispecific antibody;(b) transfecting the expression vector into a host cell by a geneticengineering method; (c) culturing the host cell under conditions thatallow the bispecific antibody to be generated; and (d) separating andpurifying the generated bispecific antibody;

The expression vector in step (a) is one or more selected from plasmids,bacteria, and viruses, and preferably the expression vector is apCDNA3.1 vector;

The host cell into which the constructed vector is transfected by thegenetic engineering method in step (b) includes a prokaryotic cell, ayeast or a mammalian cell, such as a CHO cell, an NS0 cell or anothermammalian cell, preferably a CHO cell.

The bispecific antibody is separated and purified in step (d) by aconventional immunoglobulin purification method comprising protein Aaffinity chromatography and ion exchange, hydrophobic chromatography ormolecular sieve.

In a seventh aspect of the present disclosure, use of the bispecificantibody in the preparation of a medicament for the treatment,prevention or alleviation of tumor is provided; examples of the cancerinclude, but are not limited to, mesothelioma, squamous cell carcinoma,myeloma, osteosarcoma, glioblastoma, neuroglioma, malignant epithelialtumours, adenocarcinoma, melanoma, sarcoma, acute and chronic leukemia,lymphoma and meningioma, Hodgkin's lymphoma, Sezary syndrome, multiplemyeloma, lung cancer, non-small cell lung cancer, small cell lungcancer, laryngeal cancer, breast cancer, head and neck cancer, bladdercancer, uterine cancer, skin cancer, prostate cancer, cervical cancer,vaginal cancer, gastric cancer, renal cell carcinoma, renal carcinoma,pancreatic cancer, colorectal cancer, endometrial carcinoma, esophagealcarcinoma, hepatobiliary cancer, bone cancer, skin cancer and bloodcancer, and carcinoma of nasal cavity and sinus, nasopharyngealcarcinoma, oral cancer, oropharyngeal cancer, laryngeal cancer,sublaryngeal cancer, salivary cancer, mediastinal cancer, stomachcancer, small intestine cancer, colon cancer, cancer of rectum and analregions, ureter cancer, urethral cancer, carcinoma of penis, testicularcancer, vulva cancer, cancer of endocrine system, cancer of centralnervous system, and plasmocytoma.

In an eighth aspect of the present disclosure, provided is thebispecific antibody for use in a method for enhancing or stimulating animmune response or function, comprising administering to apatient/subject individual a therapeutically effective amount of thebispecific antibody.

In a ninth aspect of the present disclosure, provided is the bispecificantibody for use in a method for treating, delaying development of, orreducing/inhibiting recurrence of a tumor, comprising: giving oradministering an effective amount of the bispecific antibody to anindividual suffering from the foregoing disease or disorder; examples ofthe tumor include, but are not limited to, mesothelioma, squamous cellcarcinoma, myeloma, osteosarcoma, glioblastoma, neuroglioma, malignantepithelial tumours, adenocarcinoma, melanoma, sarcoma, acute and chronicleukemia, lymphoma and meningioma, Hodgkin's lymphoma, Sezary syndrome,multiple myeloma, lung cancer, non-small cell lung cancer, small celllung cancer, laryngeal cancer, breast cancer, head and neck cancer,bladder cancer, uterine cancer, skin cancer, prostate cancer, cervicalcancer, vaginal cancer, gastric cancer, renal cell carcinoma, renalcarcinoma, pancreatic cancer, colorectal cancer, endometrial carcinoma,esophageal carcinoma, hepatobiliary cancer, bone cancer, skin cancer andblood cancer, and carcinoma of nasal cavity and sinus, nasopharyngealcarcinoma, oral cancer, oropharyngeal cancer, laryngeal cancer,sublaryngeal cancer, salivary cancer, mediastinal cancer, cervicalcancer, small intestine cancer, colon cancer, cancer of rectum and analregions, ureter cancer, carcinoma of urethral cancer, carcinoma ofpenis, testicular cancer, vulva cancer, cancer of endocrine system,cancer of central nervous system, and plasmocytoma.

The technical solutions provided by the present disclosure havebeneficial effects summarized as follows:

1. The bispecific antibody provided by the present disclosure includesanti-TAA scFv located at the N-terminus of the bispecific antibody andhaving changed spatial conformation, so that the bispecific antibody hasreduced binding ability to TAA under some conditions, especially thatthe bispecific antibody is difficult to bind to normal cells with weakexpression or low expression of TAAs, thereby exhibiting reducednon-specific killing effect. However, the binding specificity to cellswith over-expression or high expression of TAA is not significantlyreduced, and the bispecific antibody exhibits a good killing effect invivo. It can be known that when a target antigen is merely expressed ontumor cells or the bispecific antibody of the present disclosurespecifically binds to tumor cells over-expressing the target antigen,immune effector cells are activated restrictively and merely in targetcell tissues, which can minimize the non-specific killing of thebispecific antibody on the normal cells and the accompanying release ofcytokines and reduce the toxic side effects of the bispecific antibodyin clinical treatment.

2. The anti-CD3 scFv selected by the bispecific antibody provided by thepresent disclosure specifically binds to effector cells with a weakbinding affinity (EC₅₀ value greater than about 50 nM, or greater than100 nM, or greater than 300 nM, or greater than 500 nM). In addition,the anti-CD3 scFv is embedded between the anti-TAA scFv and Fc, and theCTP rigid peptide contained in the linker peptide L3 at the N-terminusand the Fc fragment located at its C-terminus partially “cover” or“shield” the antigen-binding domain of the anti-CD3 scFv. Such sterichindrance effect makes the anti-CD3 scFv bind to CD3 with a weakerbinding affinity (for example, greater than 1 μM), which reduces itsability to activate and stimulate T cells, limits the excessive releaseof cytokines, and provides higher safety. In addition, the anti-CD3 scFvused in the present disclosure can bind to CD3 native antigens fromhumans and cynomolgus monkeys and/or rhesus monkeys at the same time, sothat no alternative molecule needs to be constructed for preclinicaltoxicology evaluation and the effective dose, toxic dose and toxic sideeffects obtained are more objective and accurate and can be directlyconverted into a clinical dose to reduce the risk of clinical studies.Further, the bispecific antibody provided by the present disclosurecreatively adopts a divalent anti-CD3 scFv, which avoids an asymmetricstructure of a heterodimer (including a monovalent anti-CD3 scFv)commonly used in the existing art in terms of the configuration designof the bispecific antibody and solves the problem of heavy chainmismatches, thereby simplifying downstream purification steps. Moreover,unexpectedly, the non-specific binding of the anti-CD3 scFv to T cellsis not observed in an in vitro cell binding assay, and the degree ofcell activation (the release of cytokines such as IL-2) is controlledwithin a safe and effective range. That is, the bivalent anti-CD3 scFvstructure used in the present disclosure has not induced theover-activation of T cells in a non-antigen-dependent manner, whereasfor other bispecific antibodies including bivalent anti-CD3 domains, theuncontrollable over-activation of T cells is common and thus anti-CD3bispecific antibodies are generally designed to avoid the introductionof a bivalent anti-CD3 structure.

3. The modified Fc fragment included in the bispecific antibody providedby the present disclosure has no ability of binding to FcγR, avoidingthe systemic activation of T cells mediated by FcγR and allowing immuneeffector cells to be activated restrictively and merely in target celltissues.

4. The bispecific antibody provided by the present disclosure ishomodimeric without mismatches of heavy chains and light chains. Thebispecific antibody is produced by a stable downstream process andpurified by simple and efficient steps, with a homogeneous expressionproduct and significantly improved physicochemical and in vivostability.

5. The bispecific antibody provided by the present disclosure has arelatively long in vivo circulating half-life due to the inclusion ofthe Fc fragment. Pharmacokinetic tests show that the in vivo circulatinghalf-life in mice and cynomolgus monkeys are about 40 hours and 8 hours,respectively, which will greatly reduce a clinical administrationfrequency.

DETAILED DESCRIPTION Abbreviations and Definitions

-   Her2 Human epidermal growth factor receptor 2-   BiAb Bispecific antibody-   CDR Complementarity determining region in a variable region of an    immunoglobulin, defined by a Kabat numbering system-   EC₅₀ A concentration at which 50% efficacy or binding is generated-   ELISA Enzyme-linked immunosorbent assay-   FR Framework region of an antibody: a variable region of an    immunoglobulin excluding CDRs-   HRP Horseradish peroxidase-   IL-2 Interleukin 2-   IFN Interferon-   IC₅₀ A concentration at which 50% inhibition is generated-   IgG Immunoglobulin G-   Kabat Immunoglobulin comparison and numbering system advocated by    Elvin A Kabat-   mAb Monoclonal antibody-   PCR Polymerase chain reaction-   V region IgG chain fragment whose sequence is variable for different    antibodies; the V region extends to Kabat residue 109 of a light    chain and residue 113 of a heavy chain.-   VH Heavy chain variable region of an immunoglobulin-   VK κ light chain variable region of an immunoglobulin-   K_(D) Equilibrium dissociation constant-   k_(a) Association rate constant-   k_(d) Dissociation rate constant

In the present disclosure, unless otherwise specified, the scientificand technical terms used herein have meanings generally understood bythose skilled in the art. The antibody or fragments thereof used in thepresent disclosure may be further modified using conventional techniquesknown in the art alone or in combination, such as amino acid deletion,insertion, substitution, addition, and/or recombination and/or othermodification methods. A method for introducing such modifications into aDNA sequence of an antibody according to the amino acid sequence of theantibody is well known to those skilled in the art. See, for example,Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory (1989), N.Y. Such modifications are preferably performed at anucleic acid level. Meanwhile, for a better understanding of the presentdisclosure, the definitions and explanations of related terms areprovided below.

“CD19”, as known as Cluster of Differentiation 19 polypeptide, is asingle channel Type I transmembrane glycoprotein with two C2-set Ig-like(immunoglobulin-like) domains and a relatively large cytoplasmic tailthat is highly conserved among mammalian species. CD19 is expressed inalmost all B lineage cells and follicular cells and plays anindispensable role in B lymphocyte differentiation. It works with CD21,CD81, and CD225 as the B cell key co-receptor. Therefore, CD19 acts as abiomarker for B lymphocyte development, B-cell lymphoma diagnosis, andB-lymphoblastic leukemia diagnosis. In addition, mutations in CD19 areassociated with severe immunodeficiency syndromes. Indications for CD19targets also include other related diseases or disorders found in theexisting art or about to be discovered in the future. The term alsoincludes any variants, isotypes, derivatives, and species homologues ofCD19 that are naturally expressed by cells including tumor cells orexpressed by cells transfected with CD19 genes or cDNA.

“CD20”, as known as Cluster of Differentiation 20 polypeptide, belongsto a four transmembrane protein and is a B lymphocyte surface-specificdifferentiation antigen. It is expressed on more than 90% of B lymphomacells and normal B lymphocytes but not expressed on hematopoietic stemcells, primary B lymphocytes, normal blood cells, and other tissues, andexhibits no significant internalization and shedding or antigenapoptosis and change after binding to antibodies, which thus may be usedas an ideal target for treating B cell lymphoma. CD20 exerts ananti-tumor effect mainly through the action of ADCC, CDC, etc. In recentyears, indications for CD20 targets have been developed, including, forexample, autoimmune diseases (including multiple sclerosis (MS), Crohn'sdisease (CD)), inflammatory diseases (e.g., ulcerative colitis (UC)),and the like. Indications for CD20 targets also include other relateddiseases or disorders found in the existing art or about to bediscovered in the future. The term also includes any variants, isotypes,derivatives, and species homologues of CD20 that are naturally expressedby cells including tumor cells or expressed by cells transfected withCD20 genes or cDNA.

“CD22”, as known as Cluster of Differentiation 22 polypeptide, has an Igdomain and is a transmembrane receptor on the surface of mature B cells.In humans, CD22 primarily inhibits the over-activation of B cell surfacereceptors and reduces the risk of developing autoimmune diseases (e.g.,systemic lupus erythematosus). Indications associated with CD22 include,for example, B-cell lymphoma, acute and chronic leukemia, and disordersassociated with other B-cell dysplasia and B-cell dependent autoimmunediseases. Indications for CD22 targets also include other relateddiseases or disorders found in the existing art or about to bediscovered in the future. The term also includes any variants, isotypes,derivatives, and species homologues of CD22 that are naturally expressedby cells including tumor cells or expressed by cells transfected withCD22 genes or cDNA.

“CD30”, is a member of the tumor necrosis factor (TNF) receptorsuperfamily, belongs to the Type I transmembrane glycoprotein, and isphysiologically expressed by activated T and B lymphocytes. CD30 ismainly expressed in tumors originate from lymph, such as all Hodgkin'slymphoma (HL), some certain B-cell lymphomas, some certain T-celllymphomas and NK-cell lymphomas, is low-expressed on the surface ofT-cells and B-cells activated in a non-pathological state, and is notexpressed in normal cells, and therefore may be used as a correspondingtumor marker and an indicator of disease diagnosis. Indications for CD30targets also include other related diseases or disorders found in theexisting art or about to be discovered in the future. The term alsoincludes any variants, isotypes, derivatives, and species homologues ofCD30 that are naturally expressed by cells including tumor cells orexpressed by cells transfected with CD30 genes or cDNA.

“EpCAM (epithelial cell adhesion molecule)” is a transmembraneglycoprotein and is one of the earliest TAAs found in colon cancer.EpCAM is overexpressed to varying degrees in most human tumors,including, for example, lung cancer, esophageal cancer, gastric cancer,breast cancer, colorectal cancer, liver cancer, prostate cancer, andovarian cancer, and is closely related to tumor diagnosis and prognosis.In addition, the overexpression of EpCAM has been developed and appliedin clinical trials of EpCAM antibodies and tumor-associated vaccines.Indications for EpCAM targets also include other related diseases ordisorders found in the existing art or about to be discovered in thefuture. The term also includes any variants, isotypes, derivatives, andspecies homologues of EpCAM that are naturally expressed by cellsincluding tumor cells or expressed by cells transfected with EpCAM genesor cDNA.

“CEA (carcinoembryonic antigen)” is an acid glycoprotein. It is anantigen on the surface of tumor cells, has the properties of humanembryonic antigen and widely exists in the digestive system cancersoriginated from endoderm, including gastrointestinal cancer, livercancer, pancreatic cancer, and may also exist in small cell lung cancer,breast cancer, medullary thyroid cancer and carcinoid tumor. Therefore,it can be used as a broad-spectrum tumor marker for the diagnosis andtreatment of various tumors. Indications for CEA targets also includeother related diseases or disorders found in the existing art or aboutto be discovered in the future. The term also includes any variants,isotypes, derivatives, and species homologues of CEA that are naturallyexpressed by cells including tumor cells or expressed by cellstransfected with CEA genes or cDNA.

“Her2 (human epidermal growth factor receptor 2)” is a member of thehuman epidermal growth factor receptor family. The occurrence,development and severity of various tumors are closely related to theactivity of Her2. In addition to gene mutations or amplification, theup-regulation of Her2 may also activate two downstream signalingpathways, trigger a series of cascade reactions, promote unlimited cellproliferation, and ultimately lead to cancer. In addition, Her2 mayinitiate multiple metastasis-related mechanisms to increase tumor cellmetastasis ability. The amplification or overexpression of Her2 genesoccurs in various tumors such as breast cancer, ovarian cancer, gastriccancer, lung adenocarcinoma, prostate cancer, and invasive uterinecancer. Indications targeting Her2 include other related diseases ordisorders found in the existing art or about to be discovered in thefuture. The term also includes any variants, isotypes, derivatives, andspecies homologues of Her2 that are naturally expressed by cellsincluding tumor cells or expressed by cells transfected with Her2 genesor cDNA. Species homologues include rhesus monkey Her2.

“EGFR (epidermal growth factor receptor)” is a member of the epidermalgrowth factor receptor family. It is widely distributed on the surfaceof mammalian epithelial cells, fibroblasts, glial cells, keratinocytesand other cells, and is associated with the proliferation of tumorcells, angiogenesis, tumor invasion, tumor metastasis and the inhibitionof apoptosis. Mutations or overexpression of EGFR generally lead totumors, and high or abnormal expression of EGFR is found in varioussolid tumors including glial cells, renal carcinoma, lung cancer,prostate cancer, pancreatic cancer, breast cancer, and tumors in othertissues. Indications for EGFR targets also include other relateddiseases or disorders found in the existing art or about to bediscovered in the future. The term also includes any variants, isotypes,derivatives, and species homologues of EGFR that are naturally expressedby cells including tumor cells or expressed by cells transfected withEGFR genes or cDNA.

“GPC-3 (glypican-3)” is a member of the glypican family, is highlyexpressed in most embryonic tissues, and is an inhibitor of cellproliferation. The lack of GPC-3 may lead to simpson-golabi-behmelsyndrome (SGBS). It is overexpressed in early hepatocellular carcinoma(HCC) tissues and is associated with various cancers such as HCC,melanoma, ovarian cancer, and prostate cancer. In addition, GPC-3 issilenced in malignant tumors such as malignant mesothelioma, breastcancer, lung cancer, gastric cancer and ovarian cell carcinoma, but notexpressed or low expressed in normal tissues, and thus can be used as abiomarker for the treatment and diagnosis of various tumors. Indicationsfor GPC-3 targets also include other related diseases or disorders foundin the existing art or about to be discovered in the future. The termalso includes any variants, isotypes, derivatives, and specieshomologues of GPC-3 that are naturally expressed by cells includingtumor cells or expressed by cells transfected with GPC-3 genes or cDNA.

“Mesothelin” belongs to the mesothelin family and is apre-pro-megakaryocyte potentiating factor. It can be proteolyticallycleaved, with a furin invertase protein, into two chains:megakaryocyte-potentiating factor (MPF) and mesothelin. Mesothelin is atumor differentiation antigen and usually found on mesothelial cellslining the pleura, peritoneum and pericardium. Mesothelin isoverexpressed and immunogenic in a variety of tumors such asmesothelioma, ovarian cancer, lung cancer, and pancreatic cancer, andtherefore can be used as a tumor marker or antigenic target fortherapeutic cancer vaccines. Indications targeting Mesothelin includeother related diseases or disorders found in the existing art or aboutto be discovered in the future. The term also includes any variants,isotypes, derivatives, and species homologues of Mesothelin that arenaturally expressed by cells including tumor cells or expressed by cellstransfected with Mesothelin genes or cDNA.

“Mucin1 (cell surface associated mucin protein 1)” is a member of themucin protein family, and is expressed on the apical surface ofepithelial cells in tissue organs including lungs, breast, stomach andpancreas. The overexpression, aberrant intracellular localization, andchanges in glycosylation of Mucin1 are associated with cancer including,but not limited to, colon cancer, breast cancer, ovarian cancer, lungcancer, and pancreatic cancer. Using immunohistochemistry, Mucin1 can beidentified in a wide range of secretory epithelial cells, mesenchymaltumors (e.g., synovial sarcoma and granulosa cell tumor of ovary), andtheir neoplastic equivalents. Moreover, Mucin1 can be used todistinguish mesothelioma (in which it is restricted to the cellmembranes and associated microvilli), from adenocarcinoma, in which itis diffusely spread through the cytoplasm. Therefore, Mucin1 can be usedto diagnose and treat the above related diseases or disorders and otherrelated diseases or disorders found in the existing art or about to bediscovered in the future. The term also includes any variants, isotypes,and species homologues of Mucin1 that are naturally expressed by cellsincluding tumor cells or expressed by cells transfected with Mucin1genes or cDNA.

“CA125 (carbohydrate antigen 125)” is an ovarian cancer-associatedantigen that originates from fetal coelomic epithelial tissue and iswidely distributed on the surface of mesothelial cells such as pleura,pericardium, peritoneum, endometrium, genital tract and amnioticmembrane. CA-125 levels in serum are significantly elevated whenmalignant lesions occur in these sites or these sites are stimulated byinflammation. As the most studied ovarian cancer marker, CA125 has beenreported in the study of early screening, diagnosis, treatment andprognosis of ovarian cancer. CA125 levels in the puncture fluid ofbenign cystic tumor and malignant cystic epithelioma of ovarian cancerare significantly elevated. CA125 serum levels are also elevated ingastrointestinal malignant tumors (e.g., pancreatic cancer, livercancer, gastric cancer, and bowel cancer), chronic pancreatitis, chronichepatitis, liver cirrhosis, lung adenocarcinoma, pelvic inflammatorydisease, and endometriosis. Given the research of CA125 in a variety ofcancers and inflammation, CA125 can be widely used in screening,diagnosis and treatment of the above related diseases. Indications forCA125 targets also include other related diseases or disorders found inthe existing art or about to be discovered in the future. The term alsoincludes any variants, isotypes, and species homologues of CA125 thatare naturally expressed by cells including tumor cells or expressed bycells transfected with CA125 genes or cDNA.

“BCMA (B-cell maturation antigen)” is a member of the tumor necrosisfactor receptor superfamily. BCMA is preferentially expressed in matureB lymphocytes and is also expressed on the surface of plasmablasts(i.e., plasma cell precursors) and plasma cells. RNAs of BCMA aredetected in the spleen, lymph node, thymus, adrenal gland and liver, andthe level of BCMA mRNA in multiple B-cell lines also increases aftermaturation. BCMA is associated with a variety of diseases such asleukemia, lymphoma (e.g., Hodgkin's lymphoma), multiple myeloma, andautoimmune diseases (e.g., systemic lupus erythematosus), and thereforecan be used as a potential target for related B-cell diseases.Indications for BCMA targets also include other related diseases ordisorders found in the existing art or about to be discovered in thefuture. The term also includes any variants, isotypes, and specieshomologues of BCMA that are naturally expressed by cells including tumorcells or expressed by cells transfected with BCMA genes or cDNA.

CD3 molecule is an important differentiation antigen on the T cellmembrane and a characteristic marker of mature T cells. It is composedof six peptide chains, and these chains are associated with the T cellantigen receptor (TCR) with a non-covalent bond to constitute a TCR-CD3complex. CD3 molecule not only participates in the intracytoplasmicassembly of the TCR-CD3 complex but also transmits antigen stimulationsignals through the immunoreceptor tyrosine-based activation motif(ITAM) of the cytoplasmic regions of polypeptide chains. The mainfunctions of CD3 molecule are to stabilize TCR structure and transmit Tcell activation signal. When TCR specifically recognizes and binds tothe antigen, CD3 is involved in signal transduction into T cellcytoplasm as the first signal to induce T cell activation and plays avery important role in T cell antigen recognition and immune responsegeneration.

“CD3” refers to a part of a T-cell receptor complex and consists ofthree different chains CD3ε, CD3δ and CD3γ. CD3 is clustered on T cellsby, for example, being immobilized by an anti-CD3 antibody, leading tothe activation of T cells, which is similar to T cell receptor-mediatedactivation but independent of the specificity of TCR clones. Mostanti-CD3 antibodies recognize the chain CD3F. The second functionaldomain that specifically recognizes the T cell surface receptor CD3 inthe present disclosure is not specifically limited as long as it canspecifically recognize CD3, for example, but not limited to, CD3antigens mentioned in the following patents: U.S. Pat. Nos. 7,994,289;6,750,325; 6,706,265; 5,968,509; 8,076,459; 7,728,114; and U.S.20100183615. Preferably, the antibody against human CD3 used in thepresent disclosure is cross-reactive with cynomolgus monkeys and/orrhesus monkeys, for example, but not limited to, CD3 antigens mentionedin the following patents: WO 2016130726, U.S. 20050176028, WO2007042261, or WO 2008119565. The term also includes any variants,isotypes, derivatives, and species homologues of CD3 that are naturallyexpressed by cells or expressed by cells transfected with a gene or cDNAencoding the preceding chains.

The term “hypervariable region”, “CDR” or “complementarity determiningregion” refers to amino acid residues of an antibody, which areresponsible for antigen binding, and is a discontinuous amino acidsequence. CDR sequences are amino acid residues in the variable regionthat may be defined by the IMGT, Kabat, Chothia or AbM method oridentified by any CDR sequence determination method well known in theart. For example, the hypervariable region includes the following aminoacid residues: amino acid residues from a “complementarity determiningregion” or “CDR” defined by sequence comparison, for example, residuesat positions 24-34 (L1), 50-56 (L2) and 89-97 (L3) in a light chainvariable domain and residues at positions 31-35 (H1), 50-65 (H2) and95-102 (H3) in a heavy chain variable domain (see Kabat et al., 1991,Sequences of Proteins of Immunological Interest (5th edition), PublicHealth Service, National Institutes of Health, Bethesda, Md.), and/oramino acid residues from a “hypervariable loop” (HVL) defined accordingto the structure, for example, residues at positions 26-32 (L1), 50-52(L2) and 91-96 (L3) in the light chain variable domain and residues atpositions 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chainvariable domain (see Chothia and Leskl, J. Mol Biol, 196: 901-917,1987). “Framework” residues or “FR” residues refer to variable domainresidues other than the hypervariable region residues as defined in thepresent disclosure. In some embodiments, the antibody or theantigen-binding fragment thereof in the present disclosure is preferablydetermined through the Kabat, Chothia or IMGT numbering system. Thoseskilled in the art may explicitly assign each system to any variabledomain sequence without relying on any experimental data beyond thesequence itself. For example, the Kabat residue numbering method of agiven antibody may be determined by comparing the sequence of the givenantibody to each “standard” numbered sequence. Based on the numbers ofthe sequences provided herein, the numbering scheme of determining anyvariable region sequence in the sequence table is entirely within theconventional technical scope of those skilled in the art.

The term “single-chain Fv antibody” (or “scFv antibody”) refers to anantibody fragment comprising VH and VL domains of an antibody. It is afusion protein of the variable regions of the heavy (VH) and lightchains (VL) connected with a linker. The linker enables these twodomains to be cross-linked to form an antigen-binding site, and thesequence of the linker generally consists of a flexible peptide, forexample, but not limited to, G₂(GGGGS)₃. The size of scFv is generally1/6 of an intact antibody. The single-chain antibody is preferably anamino acid chain sequence encoded by a nucleotide chain. For the reviewof scFv, reference may be made to Pluckthun (1994), The Pharmacology ofMonoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore,Springer-Verlag, New York, pages 269-315. Reference may also be made toInternational Patent Application Publication No. WO 88/01649 and U.S.Pat. Nos. 4,946,778 and 5,260,203.

The term “Fab fragment” consists of a light chain and a CH1 and avariable domain of each of a heavy chain. The heavy chain of the Fabmolecule cannot form a disulfide bond with another heavy chain molecule.The size of “Fab antibody” is 1/3 of an intact antibody, and “Fabantibody” includes only one antigen-binding site.

The term “Fab′ fragment” contains a light chain, and a VH domain and aCH1 domain of a heavy chain, and a constant region between CH1 and CH2domains.

The term “F(ab′)₂ fragment” contains two light chains, VH domains andCH1 domains of two heavy chains, and constant regions between CH1 andCH2 domains, so that an inter-chain disulfide bond is formed between thetwo heavy chains. Therefore, the F(ab′)₂ fragment is composed of twoFab′ fragments held together by the disulfide bond between the two heavychains.

The term “Fc” region refers to the antibody heavy chain constant regionfragment, including at least a hinge region and CH2 and CH3 domains.

The term “Fv region” includes variable regions from the heavy chain andthe light chain but lacks the constant regions, and is the minimumfragment containing a complete antigen recognition and binding site.

The term “antibody fragment” and “antigen-binding fragment” refers to anantigen-binding fragment of the antibody that retains a specific bindingability to an antigen (e.g., Her2), as well as antibody analogs. Itgenerally includes at least part of an antigen-binding region or avariable region of a parental antibody. The antibody fragment retains atleast part of the binding specificity of the parental antibody.Generally, when the activity is represented in moles, the antibodyfragment retains at least 10% of parental binding activity. Preferably,the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or100% of the binding affinity of the parental antibody to a target. Theantibody fragments include, but are not limited to, Fab fragments, Fab′fragments, F(ab′)2 fragments, Fv fragments, Fd fragments,complementarity determining region (CDR) fragments, disulfide-stabilizedvariable fragments (dsFv); linear antibodies, single-chain antibodies(e.g., scFv monoclonal antibodies) (technology from Genmab), bivalentsingle-chain antibodies, single-chain phage antibodies, single domainantibodies (e.g., VH domain antibodies), domain antibodies (technologyfrom AbIynx); multispecific antibodies formed from antibody fragments(e.g., triabodies and tetrabodies); and engineered antibodies such aschimeric antibodies (e.g., humanized mouse antibodies) andheteroconjugate antibodies. These antibody fragments can be obtainedusing any conventional technologies known to those skilled in the art,and the utility of these fragments can be screened in the same way asthe intact antibody.

The term “linker peptide” refers to a peptide linking two polypeptides,wherein the linker peptide may be two immunoglobulin variable regions orone variable region. The length of the linker peptide may be 0 to 30amino acids or 0 to 40 amino acids. In some embodiments, the linkerpeptide may be in the length of 0 to 25, 0 to 20, or 0 to 18 aminoacids. In some embodiments, the linker peptide may be a peptide havingno more than 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids. In otherembodiments, the linker peptide may include 0 to 25, 5 to 15, 10 to 20,15 to 20, 20 to 30, or 30 to 40 amino acids. In other embodiments, thelinker peptide may have about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30amino acids. The linker peptide is known to those skilled in the art.The linker peptide may be prepared by any method in the art. Forexample, the linker peptide may be originated from synthesis.

The term “heavy chain constant region” includes an amino acid sequencefrom the immunoglobulin heavy chain. The polypeptide comprising theheavy chain constant region includes at least one of: a CH1 domain, ahinge domain (e.g., an upper hinge region, an intermediate hinge region,and/or a lower hinge region), a CH2 domain, a CH3 domain, or a variantor fragment thereof. For example, the antigen-binding polypeptide usedherein may include a polypeptide chain having a CH1 domain; apolypeptide having a CH1 domain, at least part of a hinge domain, and aCH2 domain; a polypeptide chain having a CH1 domain and a CH3 domain; apolypeptide chain having a CH1 domain, at least part of a hinge domain,and a CH3 domain; or a polypeptide chain having a CH1 domain, at leastpart of a hinge domain, a CH2 domain, and a CH3 domain. In anotherembodiment, the polypeptide of the present application includes apolypeptide chain having a CH3 domain. In addition, the antibody used inthe present application may lack at least part of a CH2 domain (e.g.,all or part of a CH2 domain). As described above, it is appreciated bythose of ordinary skill in the art that heavy chain constant regions maybe modified such that they differ in amino acid sequence from naturallyimmunoglobulin molecules.

The term “light chain constant region” includes an amino acid sequencefrom the antibody light chain. Preferably, the light chain constantregion includes at least one of a constant kappa domain and a constantlambda domain.

The term “VH domain” includes an amino-terminal variable domain of theimmunoglobulin heavy chain, while the term “CH1 domain” includes a first(mostly amino-terminal) constant region of the immunoglobulin heavychain. The CH1 domain is adjacent to the VH domain and is theamino-terminal of the hinge region of the immunoglobulin heavy chainmolecule.

“Binding” defines the affinity interaction between a specific epitope onan antigen and its corresponding antibody and is generally understood as“specific recognition.” “Specific recognition” means that the bispecificantibody of the present disclosure does not or substantially does nothave cross-reaction with any polypeptide other than the target antigen.The degree of specificity may be determined by immunological techniques,including, but not limited to, immunoblotting, immunoaffinitychromatography, and flow cytometry. In the present disclosure, thespecific recognition is preferably determined by flow cytometry, and thecriteria for the specific recognition in particular cases can be judgedby those of ordinary skill in the art according to the general knowledgeof the art which he/she knows.

The term “in vivo half-life” refers to the biological half-life of thepolypeptide of interest in the circulation of a given animal and isexpressed as the time it takes to clear half of the amount present inthe circulation of the animal from the circulation and/or other tissuesin the animal.

The term “identity” refers to the matching of sequences between twopolypeptides or between two nucleic acids. When a certain position ineach of two sequences for comparison is occupied by the same base groupor amino acid monomer subunit (e.g., a certain position in each of thetwo DNA molecules is occupied by adenine, or a certain position in eachof the two polypeptides is occupied by lysine), then the molecules areidentical at that position. The “percentage identity” between twosequences is a function of the number of matching positions of the twosequences divided by the number of positions to be compared and thenmultiplied by 100. For example, if 6 of the 10 positions in twosequences are matched, the identity between the two sequences is 60%.For example, DNA sequences CTGACT and CAGGTT have a total identity of50% (three of a total of six positions are matched). Generally, thecomparison is made when two sequences are aligned to produce maximumidentity. Such alignment may be implemented, for example, through acomputer program, such as an Align program (DNAstar, Inc.) toconveniently perform the method described by Needleman et al. Mol.Biol., 48: 443-453. The percentage identity between the two amino acidsequences may also be determined by using the algorithm proposed by E.Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17) which has beenincorporated into ALIGN program (version 2), using the PAM 120 weightresidue table with a gap length penalty score of 12 and a gap penaltyscore of 4. In addition, the percentage identity between the two aminoacid sequences may also be determined by using the algorithm proposed byNeedleman and Wunsch (J. Mol. Biol., 48: 444-453) which has beenincorporated into the GAP program in the GCG software package (availableon www.gcg.com), using Blossum 62 matrix or PAM 250 matrix with a gapweight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5or 6.

The term “Fc region” or “Fc fragment” refers to the C-terminal regionsof the immunoglobulin heavy chain, which includes at least part of ahinge region, a CH2 domain and a CH3 domain. It mediates the binding ofimmunoglobulins to host tissues or factors, including the binding ofimmunoglobulins to Fc receptors located on various cells (e.g., effectorcells) of the immune system or the binding of immunoglobulins to thefirst component (C1q) of the classical complement system. It includesthe native sequence Fc region and the variant Fc region.

Generally, the human IgG heavy chain Fc region is a segment from theamino acid residue at the position Cys226 or Pro230 of the human IgGheavy chain Fc region to the carboxy terminus, but its boundaries mayvary. The C-terminal lysine of the Fc region (residue 447, according tothe EU numbering system) may or may not be present. Fc may also refer tothis region in isolation, or in the case of a protein polypeptidecomprising Fc, for example, “binding protein comprising an Fc region”,and is also referred to as “Fc fusion protein” (e.g., antibody orimmunoadhesin). The native Fc region of the antibody of the presentdisclosure includes mammalian (e.g., human) IgG1, IgG2 (IgG2A, IgG2B),IgG3, and IgG4. Among human IgG1 Fc regions, at least two allotypes areknown. In some embodiments, there is a single amino acid substitution,insertion, and/or deletion of about 10 amino acids per 100 amino acidsin the amino acid sequences of two Fc polypeptide chains relative to thesequence of the amino acid sequence of the mammalian Fc polypeptide. Insome embodiments, the difference may be changes in Fc that extend thehalf-life, changes that increase FcRn binding, changes that inhibit Fc7receptor (FcγR) binding, and/or changes that decrease or remove ADCC andCDC.

The term “Fc receptor” or “FcR” refers to a receptor that binds to theFc region of an immunoglobulin. FcR may be a native sequence human FcR,or may be an FcR that binds to the IgG antibody (a y receptor), as wellas allelic variants and alternatively spliced forms of these receptors.The FcγR family is composed of three activating receptors (FcγRI,FcγRIII and FcγRIV in mice; FcγRIA, FcγRIIA and FcγRIIIA in humans) andone inhibitory receptor (FcγRIIb or equivalent FcγRIIB). The FcγRIIreceptor includes FcγRIIA (“activating receptor”) and FcγRIIB(“inhibitory receptor”) which have similar amino acid sequences. Thecytoplasmic domain of FcγRIIA includes an immunoreceptor tyrosine-basedactivation motif (ITAM). The cytoplasmic domain of FcγRIIB contains animmunoreceptor tyrosine-based inhibitory motif (ITIM) (see M. Annu. Rev.Immunol., 15: 203-234 (1997)). Most native effector cell typesco-express one or more activating FcγRs and inhibitory FcγRIIbs, whileNK cells selectively express one activating Fc receptor (FcγRIII in miceand FcγRIIIA in humans) but do not express inhibitory FcγRIIb in miceand humans. Human IgG1 binds to most human Fc receptors and isconsidered equivalent to murine IgG2a in terms of the type of activatingFc receptor to which Human IgG1 binds. The term “FcR” herein coversother FcRs, including those which will be identified in the future.Methods of measuring the binding to FcRn are known (see, e.g., Ghetie Vet al., Immunol Today, 18: 592-8, 1997); Ghetie V et al., NatureBiotechnology, 15: 637-40, 1997)). The in vivo binding and serumhalf-life of the human FcRn high-affinity binding polypeptide to FcRncan be determined, for example, in transgenic mice expressing human FcRnor transfected human cell lines. The term “Fc receptor” or “FcR” alsoincludes the neonatal receptor FcRn which is responsible fortransferring maternal IgG to the fetus (Guyer R L et al., J. Immunol.,117: 587, 1976) and Kim Y J et al., J. Immunol., 24: 249, 1994)).

The term “humanized antibody” refers to a genetically engineerednon-human antibody whose amino acid sequence has been modified toincrease homology to the sequence of the human antibody. Most or all ofthe amino acids outside the CDR domain of a non-human antibody, forexample, a mouse antibody, are substituted by corresponding amino acidsfrom human immunoglobulins, while most or all of the amino acids withinone or more CDR regions are not altered. The addition, deletion,insertion, substitution or modification of small molecule amino acids ispermissible as long as they do not eliminate the ability of the antibodyto bind a particular antigen. The “humanized” antibody retains antigenspecificity similar to that of the original antibody. The origin of theCDRs is not particularly limited and may be derived from any animal. Forexample, antibodies derived from mouse antibodies, rat antibodies,rabbit antibodies, or non-human primate (e.g., cynomolgus monkeys)antibodies may be used. Examples of human frameworks useful in thepresent disclosure are KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (Kabatet al., ibid). For example, KOL and NEWM may be used for heavy chains,REI may be used for light chains, and EU, LAY, and POM may be used forboth heavy and light chains. Alternatively, human germline sequences maybe used, and these sequences are available athttp://www2.mrc-lmb.cam.ac.uk/vbase/list2.php. In the humanized antibodymolecules of the present disclosure, the receptor heavy and light chainsdo not need to be derived from the same antibody, and may, if needed,include complex chains having framework regions derived from differentchains.

The term “cytokine” generally refers to a protein that is released byone cell population and that acts as an intercellular medium on anothercell or has an autocrine effect on the cells from which the protein isproduced. Examples of such cytokines include lymphokines, monokines,interleukins (“IL”) such as IL-2, IL-6, and IL-17A-F, tumor necrosisfactors such as TNF-α and TNF-β, and other polypeptide factors such asleukemia inhibitory factor (“LIF”).

The term “immunobinding” and “immunobinding property” refers to anon-covalent interaction between an immunoglobulin molecule and anantigen (to the antigen, the immunoglobulin is specific). The strengthor affinity of the immunobinding interaction may be represented by theequilibrium dissociation constant (K_(D)) of the interaction, where thesmaller the K_(D) value, the higher the affinity. The immunobindingproperty of the selected polypeptide may be quantified using a methodknown in the art. One method relates to the measurement of rates atwhich an antigen-binding site/antigen complex is formed and dissociated.Both the “binding rate constant” (K_(a) or K_(on)) and the “dissociationrate constant” (K_(d) or K_(off)) may be calculated according to theconcentration and actual rates of association and dissociation (seeMalmqvist M et al., Nature, 361: 186-187, 1993). The ratio ofk_(d)/k_(a) is the dissociation constant K_(D) (generally see Davies etal., Annual Rev Biochem., 1990, 59: 439-473). Any effective method maybe used for measuring values of K_(D), k_(a) and k_(d).

The term “cross-reaction” refers to the ability of the antibodydescribed herein to bind to tumor-associated antigens from differentspecies. For example, the antibody described herein that binds to humanTAA may also bind to TAAs from other species (e.g., cynomolgus monkeyTAA). Cross-reactivity may be measured by detecting specific reactivitywith purified antigens in binding assays (e.g., SPR, ELISA), ordetecting the binding to cells physiologically expressing TAA or theinteraction with the function of cells physiologically expressing TAA.Examples of assays known in the art for determining the binding affinityinclude surface plasmon resonance (e.g., Biacore) or similar techniques(e.g., Kinexa or Octet).

The term “EC₅₀” refers to the maximum response of the concentration ofthe antibody or antigen-binding fragment thereof that induces a 50%response in an in vitro or in vivo assay using the antibody orantigen-binding fragment thereof, that is, half between the maximumresponse and the baseline.

“Effector cell” refers to a cell of the immune system, which expressesone or more FcRs and mediates one or more effector functions.Preferably, the cell expresses at least one type of activating Fcreceptors such as human FcγRIII and performs ADCC effector function.Examples of human leukocytes which mediate ADCC include peripheral bloodmononuclear cells (PBMCs), natural killer (NK) cells, monocytes,macrophages, neutrophils, and eosinophils. Effector cells also include,for example, T cells. They may be derived from any organism including,but not limited to, humans, mice, rats, rabbits or monkeys.

The term “effector function” refers to biological activities that can beattributed to the biological activities of the antibody Fc region (anative sequence Fc region or amino acid sequence variant Fc region) andthat vary with antibody isotypes. Examples of antibody effectorfunctions include, but are not limited to, Fc receptor binding affinity,ADCC, ADCP, CDC, downregulation of cell surface receptors (e.g., B cellreceptors), B cell activation, cytokine secretion, andhalf-life/clearance rate of antibodies and antigen-antibody complexes.Methods of altering the effector function of antibodies are known in theart, for example, the effector function of antibodies may be altered byintroducing mutations in the Fc region.

The term “antibody-dependent cell-mediated cytotoxicity (ADCC)” refersto a cytotoxic form in which Ig binds to FcRs on cytotoxic cells (e.g.,NK cells, neutrophils or macrophages) to enable these cytotoxic effectorcells to specifically bind to antigen-attached target cells and thensecret cytotoxins to kill the target cells. Methods for detecting theADCC activity of an antibody are known in the art, for example, the ADCCactivity may be detected by measuring the binding activity between ato-be-tested antibody and FcR (e.g., CD16a).

The term “antibody-dependent cell-mediated phagocytosis (ADCP)” refersto a cell-mediated reaction in which a non-specific cytotoxic activecell expressing FcγR recognizes a bound antibody on a target cell andsubsequently causes phagocytosis of the target cell.

The term “complement-dependent cytotoxicity (CDC)” refers to a cytotoxicform that activates the complement cascade by binding the complementcomponent C1q to the antibody Fc. Methods for detecting the CDC activityof an antibody are known in the art, for example, the CDC activity maybe detected by measuring the binding activity between a to-be-testedantibody and an Fc receptor (e.g., C1q).

The term “pharmaceutically acceptable carrier and/or excipient and/ordiluent” refers to a carrier and/or excipient and/or stabilizer which ispharmacologically and/or physiologically compatible with the subject andthe active ingredient and which is non-toxic to the cell or mammalexposed to such a carrier and/or excipient and/or stabilizer at thedosage and concentration employed. Examples include, but are not limitedto, pH regulators, surfactants, adjuvants, ionic strength enhancers,diluents, reagents to maintain osmotic pressure, reagents to delayabsorption, and preservatives. For example, pH adjusting agents include,but are not limited to, phosphate buffers. Surfactants include, but arenot limited to, cationic surfactant, anionic surfactant or nonionicsurfactants, for example, Tween-80. Ionic strength enhancers include,but are not limited to, sodium chloride. Preservatives include, but arenot limited to, various antibacterial reagent and antifungal reagent,such as parabens, chlorobutanol, phenol, and sorbic acid. Reagents tomaintain osmotic pressure include, but are not limited to, sugars, NaCl,and analogs thereof. Reagents to delay absorption include, but are notlimited to, monostearate and gelatin. Diluents include, but are notlimited to, water, aqueous buffers (e.g., buffered saline), alcohols,and polyols (e.g., glycerol). Preservatives include, but are not limitedto, various antibacterial reagent and antifungal reagent, such asthiomersal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, andsorbic acid. Stabilizers have the meaning as commonly understood bythose of ordinary skill in the art. Stabilizers are those capable ofstabilizing the desired activity of the active ingredient in a drug,including, but not limited to, sodium glutamate, gelatin, SPGA, sugars(e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran orglucose), amino acids (e.g., glutamic acid or glycine), proteins (e.g.,dry whey, albumin or casein), or degradation products thereof (e.g.,lactalbumin hydrolysate).

The term “effective amount” refers to an amount sufficient to obtain orat least partially obtain the desired effect. For example, aprophylactically (e.g., tumor or infection) effective amount refers toan amount sufficient to prevent, arrest, or delay the onset of a disease(e.g., tumor or infection) when used alone or used together with one ormore therapeutic agents; a therapeutically effective amount refers to anamount sufficient to cure or at least partially arrest the disease andcomplications thereof in a patient already suffering from the diseasewhen used alone or used together with one or more therapeutic agents. Itis well within the ability of those skilled in the art to determine sucheffective amounts. For example, the amount effective for therapeutic usedepends on the severity of the to-be-treated disease, the overall stateof the patient's own immune system, the general condition of the patientsuch as age, weight and sex, the mode of administration of the drug, andother treatments administered concurrently. The terms “efficacy” and“effectiveness” with respect to treatment include both pharmacologicaleffectiveness and physiological safety. The pharmacologicaleffectiveness refers to the ability of a drug to promote regression of acondition or symptom in a patient. The physiological safety refers tothe level of toxicity or other adverse physiological effects (adverseeffects) at the cellular, organ and/or organism level due to drugadministration.

“Treatment” or “therapy” on a subject refers to any type of interventionor treatment of, or administration of an active agent to, a subject forthe purpose of reversing, alleviating, ameliorating, inhibiting, slowingor preventing the occurrence, progression, progression, severity, orrecurrence of symptoms, complications, disorders or biochemicalindicators associated with a disease.

The term “T-cell receptor (TCR)” is a specific receptor present on thesurface of T cells, i.e., T lymphocytes. In vivo, the T-cell receptor ispresent as a complex of several proteins. The T-cell receptor generallyhas two separate peptide chains, typically T-cell receptors α and β(TCRα and TCRβ) chains, and in some T cells, they are T-cell receptor γand δ (TCRγ and TCRδ). The other proteins in the complex are CD3proteins: CD3εγ and CD3εδ heterodimers, most importantly, CD3ζhomodimers having six ITAMs. The ITAMs on CD3ζ can be phosphorylated byLck, which in turn recruit ZAP-70. Lck and/or ZAP-70 may alsophosphorylate tyrosine on many other molecules, particularly CD28, LAT,and SLP-76, which allows aggregation of signal transduction complexesaround these proteins.

The term “bispecific antibody” refers to the bispecific antibody of thepresent disclosure, for example, an anti-Her2 antibody or anantigen-binding fragment thereof, which may be derivatized or linked toanother functional molecule, for example, another peptide or protein(e.g., TAA, a cytokine and a cell surface receptor), to generate abispecific antibody that binds to at least two different binding sitesor target molecules. To produce the bispecific molecule of the presentdisclosure, the antibody of the present disclosure may be functionallylinked (e.g., by chemical coupling, gene fusion, non-covalent binding orother means) to one or more other binding molecules, for example,another antibody, antibody fragment, peptide or binding mimetic, toproduce the bispecific molecule. For example, the “bispecific antibody”refers to one including two variable domains or ScFv units such that theantibody obtained recognizes two different antigens. Various differentforms and uses of the bispecific antibody are known in the art (Chames Pet al., Curr. Opin. Drug Disc. Dev., 12:276, 2009; Spiess C et al., Mol.Immunol., 67: 95-106, 2015).

The term “hCG-β carboxy terminal peptide (CTP)” is a short peptide fromthe carboxy terminus of a human chorionic gonadotropin (hCG) β-subunit.Four reproduction-related polypeptide hormones, follicle-stimulatinghormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone(TSH), and human chorionic gonadotropin (hCG), each contain the samea-subunit and their respective specific P-subunits. The in vivohalf-life of hCG is significantly longer than those of the other threehormones, mainly due to the specific carboxy terminal peptide (CTP) onthe β-subunit of hCG. The CTP includes 37 amino acid residues and fourO-glycosylation sites, in which sugar side chain terminals are sialicacid residues. The electronegative highly-sialyl CTP can resist renalclearance and thus extend the half-life in vivo (Fares F A et al., ProcNatl Acad. Sci. USA, 1992, 89: 4304-4308, 1992).

The term “glycosylation” means that an oligosaccharide (a carbohydratecontaining two or more monosaccharides that are linked together, e.g., acarbohydrate containing 2 to about 12 monosaccharides that are linkedtogether) is attached to form a glycoprotein. The oligosaccharide sidechains are generally linked to the backbone of the glycoprotein via N-or O-linkages. The oligosaccharides of the antibodies disclosed hereinare generally CH2 domains linked to the Fc region as N-linkedoligosaccharides. “N-linked glycosylation” refers to carbohydrate moietyattachment to an asparagine residue of a glycoprotein chain. Forexample, the skilled artisan can recognize a single site useful forN-linked glycosylation at residue 297 of each of CH2 domains of murineIgG1, IgG2a, IgG2b and IgG3 and human IgG1, IgG2, IgG3, IgG4, IgA andIgD.

Homologous Antibody

In another aspect, amino acid sequences included in the heavy and lightchain variable regions of the antibody of the present disclosure arehomologous with amino acid sequences of the preferred antibody describedherein, and the antibody retains desired functional properties of thebispecific antibody of the present disclosure, for example, Her2×CD3bispecific antibody.

Antibody with Conservative Modifications

The term “conservative modification” is intended to mean that an aminoacid modification does not significantly affect or change the bindingcharacteristics of the antibody containing an amino acid sequence. Suchconservative modifications include amino acid substitutions, additionsand deletions. A modification may be introduced into the antibody of thepresent disclosure by using a standard technology known in the art, suchas a site-directed mutagenesis and a PCR-mediated mutagenesis. Aconservative amino acid substitution refers to the substitution of anamino acid residue with an amino acid residue with a similar side chain.Families of amino acid residues with similar side chains have beendescribed in detail in the art. These families include amino acids withbasic side chains (such as lysine, arginine and histidine), amino acidswith acidic side chains (such as aspartic acid and glutamic acid), aminoacids with uncharged polar side chains (such as glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine and tryptophan), aminoacids with non-polar side chains (such as alanine, valine, leucine,isoleucine, proline, phenylalanine and methionine), amino acids withβ-branched side chains (such as threonine, valine and isoleucine) andamino acids with aromatic side chains (such as tyrosine, phenylalanine,tryptophan and histidine). Therefore, one or more amino acid residues inthe CDR of the antibody of the present disclosure may be substitutedwith other amino acid residues from the same side chain family.

Fc Variant with Altered Binding Affinity for the Neonatal Receptor(FcRn)

“FcRn” used herein refers to a protein that binds to at least part ofthe Fc region of the IgG antibody and that is encoded by the FcRn gene.FcRn may be derived from any organism including, but not limited to,humans, mice, rats, rabbits or monkeys. The functional FcRn proteinincludes two polypeptides that often referred to as heavy and lightchains, in which the light chain is β-2-microglobulin and the heavychain is encoded by the FcRn gene.

The present disclosure relates to an antibody whose binding to FcRn isregulated (the regulation includes to increase or decrease the binding).For example, in some cases, increased binding may result in cellrecirculating antibodies, and thus extends, for example, the half-lifeof the therapeutic antibody. Sometimes, it is desirable to decrease theFcRn binding, for example, when the antibody is used as a diagnostic ortherapeutic antibody including a radiolabel. In addition, antibodiesexhibiting increased binding to FcRn and altered binding to other Fcreceptors such as Fcγ Rs may be used in the present disclosure.

The present application relates to an antibody including an amino acidmodification that regulates the binding to FcRn. Of particular interestis that at lower pH, the binding affinity for FcRn exhibits an increase,while at higher pH, the binding basically does not exhibit an alteredantibody that minimally includes the Fc region or functional variantsthereof.

Fc Variant with Enhanced Binding Affinity for the Neonatal Receptor(FcRn)

The plasma half-life of IgG depends on its binding to FcRn, where IgGgenerally binds to FcRn at a pH of 6.0 and dissociates from FcRn at a pHof 7.4 (the pH of plasma). Through studies on the binding site, abinding site of IgG to FcRn is modified to increase the binding capacityat the pH of 6.0. It has been proved that mutations of some residues ofa human Fcγ domain, which are essential to the binding to FcRn, canincrease the serum half-life. It has been reported that mutations atT250, M252, S254, T256, V308, E380, M428, and N434 (EU numbering) canincrease or decrease the binding affinity for FcRn (Roopenian et al.,Nat. Rev. Immunol., 7: 715-725, 2007). Korean Patent No. KR 10-1027427discloses trastuzumab (Herceptin, Genentech) variants with enhancedbinding affinity for FcRn, where these variants include one or moreamino acid modifications selected from 257C, 257M, 257L, 257N, 257Y,279Q, 279Y, 308F, and 308Y. Korean Patent Publication No. KR2010-0099179 provides bevacizumab (Avastin, Genentech) variants, wherethese variants exhibit an increased in vivo half-life through amino acidmodifications included in N434S, M252Y/M428L, M252Y/N434S, andM428L/N434S. In addition, Hinton et al. have found that T250Q and M428Lmutants increase the binding to FcRn threefold and sevenfold,respectively. At the same time, the mutation of two sites increases thebinding 28-fold. In rhesus monkeys, the M428L or T250QM/428 L mutantexhibits the plasma half-life increased twofold (Hinton P. R. et al., J.Immunol., 176: 346-356, 2006). For more mutational sites included in theFc variant with the enhanced binding affinity for the neonatal receptor(FcRn), see Chinese invention patent CN 201280066663.2. In addition,studies have shown that through the T250Q/M428L mutation on Fc fragmentsof five humanized antibodies, the interaction between Fc and FcRn isimproved, and in subsequent in vivo pharmacokinetic assays, thepharmacokinetic parameters of the Fc-mutation antibody are improvedcompared with the wild-type antibody through subcutaneousadministration, for example, the in vivo exposure is increased, theclearance rate is reduced, and the subcutaneous bioavailability isincreased (Datta-Mannan A et al., MAbs. Taylor & Francis, 4: 267-273,2012).

Other mutational sites capable of enhancing the affinity of the antibodyof the present disclosure for FcRn include, but are not limited to, thefollowing amino acid modifications: 226, 227, 230, 233, 239, 241, 243,246, 259, 264, 265, 267, 269, 270, 276, 284, 285, 288, 289, 290, 291,292, 294, 298, 299, 301, 302, 303, 305, 307, 309, 311, 315, 317, 320,322, 325, 327, 330, 332, 334, 335, 338, 340, 342, 343, 345, 347, 350,352, 354, 355, 356, 359, 360, 361, 362, 369, 370, 371, 375, 378, 382,383, 384, 385, 386, 387, 389, 390, 392, 393, 394, 395, 396, 397, 398,399, 400, 401, 403, 404, 408, 411, 412, 414, 415, 416, 418, 419, 420,421, 422, 424, 426, 433, 438, 439, 440, 443, 444, 445, and 446, wherethe numbers of the amino acids in the Fc region is numbers of the EUindexes in Kabat.

Fc variants with enhanced binding affinity for FcRn also include allother known amino acid modification sites as well as amino acidmodification sites that have not yet been found. In an optionalembodiment, the IgG variant can be optimized to gain increased ordecreased affinity for FcRn and increased or decreased affinity forhuman FcγR including, but not limited to, FcγRI, FcγRIIa, FcγRIIb,FcγRIIc, FcγRIIIa and FcγRIIIb, including allelic variants thereof.

Preferentially, the Fc ligand specificity of an IgG variant determinesits therapeutic application. The given IgG variant for therapeuticpurposes depends on the epitope or form of the target antigen as well asthe to-be-treated disease or indication. Enhanced FcRn binding may bemore preferred for most targets and indications because enhanced FcRnbinding may result in extended serum half-life. A relatively long serumhalf-life allows administration at relatively low frequencies and dosesduring treatment. This property may be particularly preferred when thetherapeutic agent is administered in order to respond to indicationsrequiring repeated administration. For some targets and indications, thereduced affinity for FcRn may be particularly preferred when the variantFc is required to have an increased clearance or reduced serumhalf-life, for example, when the Fc polypeptide is used as an imagingagent or a radiotherapy agent.

The affinity of the polypeptide for FcRn can be evaluated by methodswell known in the art. For example, those skilled in the art can performappropriate ELISA assays. As illustrated in Example 5.6, appropriateELISA assays enabled the comparison of the binding strengths of thevariants and parents to FcRn. At the pH of 7.0, the specific signalsdetected for the variant and the parent polypeptide are compared, if thespecific signal of the variant is at least 1.9 times weaker than thespecific signal of the parent polypeptide, this variant is a preferredvariant of the present disclosure and is more suitable for clinicalapplication.

FcRn may be derived from any organism including, but not limited to,humans, mice, rats, rabbits or monkeys.

Alterations to Inhibit FcγR Binding

As used herein, “alterations to inhibit FcγR binding” refers to one ormore insertions, deletions or substitutions in the Fc polypeptide chainthat inhibit binding of FcγRIIA, FcγRIIB and/or FcγRIIIA, in which thebinding is determined, for example, by a competitive binding assay(PerkinElmer, Waltham, Mass.). These alterations may be included in theFc polypeptide chain as part of the bispecific antibody. Morespecifically, alterations that inhibit binding of the Fcγ receptor(FcγR) include L234A, L235A, or any alteration that inhibitsglycosylation at the position N297, including any substitution at N297.In addition, along with the alterations that inhibit glycosylation ofthe position N297, additional alterations to stabilize the dimer Fcregion by establishing additional disulfide bridges are also expected.Further examples of alterations that inhibit FcγR binding include D265Aalterations in one Fc polypeptide chain and A327Q alterations in anotherFc polypeptide chain. Some of the above mutations are described, forexample, in Xu D et al., Cellular Immunol., 200: 16-26, 2000, of whichthe section about the above mutations and the activity evaluationthereof is incorporated herein by reference. The above numbers are basedon EU numbering.

For example, the Fc fragment included by the bispecific antibodyprovided by the present disclosure in the alterations that inhibit FcγRbinding exhibits reduced affinity for at least one of human FcγR (FcγRI,FcγRIIa or FcγRIIIa) or C1q, and has reduced effector cell functions orcomplement functions.

Other alterations that inhibit FcγR binding include sites andmodifications thereof that are well known in the art or may bediscovered in the future.

FcγR may be derived from any organism including, but not limited to,humans, mice, rats, rabbits or monkeys.

Fc Alterations to Extend the Half-Life

As used herein, “Fc alterations to extend the half-life” refers to analteration to extend the in vivo half-life of a protein that includes analtered Fc polypeptide in the Fc polypeptide chain as compared with thehalf-life of a protein that includes the same Fc polypeptide but doesnot include any altered but similar Fc. These alterations may beincluded in the Fc polypeptide chain as part of the bispecific antibody.Alterations T250Q, M252Y, S254T and T256E (alteration of threonine atposition 250 to glutamine; alteration of methionine at position 252 totyrosine; alteration of serine at position 254 to threonine; andalteration of threonine at position 256 to glutamic acid; where numbersare based on EU numbering) is an Fc alteration to extend half-life andmay be used jointly, alone or in any combination. These and otheralterations are described in detail in U.S. Pat. No. 7,083,784. Thesection about this alteration described in U.S. Pat. No. 7,083,784 isincorporated herein by reference.

Similarly, M428L and N434S are Fc alterations that extend the half-lifeand can be used jointly, alone or in any combination. These alterationsand other alterations are described in detail in U.S. Patent ApplicationPublication No. 2010/0234575 and U.S. Pat. No. 7,670,600. Sections ofsuch alterations described in U.S. Patent Application Publication No.2010/0234575 and U.S. Pat. No. 7,670,600 are incorporated herein byreference.

In addition, according to the meaning herein, any substitution at one ofthe following positions can be considered to be an Fc alternation thatextends the half-life: 250, 251, 252, 259, 307, 308, 332, 378, 380, 428,430, 434, and 436. Each of these alterations or a combination of thesealterations may be used to extend the half-life of the bispecificantibody described herein. Other alternations that can be used to extendthe half-life are described in detail in International Application No.PCT/US2012/070146 (Publication No. WO 2013/096221) filed Dec. 17, 2012.The section about the above alterations of this application isincorporated herein by reference.

Fc alternations that extend the half-life also include sites andmodifications thereof that are well known in the art or may bediscovered in the future.

Fc may be derived from any organism including, but not limited to,humans, mice, rats, rabbits or monkeys.

Method for Preparing a Bispecific Antibody

The bispecific antibody of the present disclosure may be prepared by anymethod known in the art. Early methods for constructing the bispecificantibody include chemical cross-linking or hybridoma heterozygosis orquadroma method (e.g., Staerz U D et al., Nature, 314: 628-31, 1985;Milstein C et al., Nature, 305: 537-540, 1983; Karpovsky B et al., J.Exp. Med., 160: 1686-1701, 1984). The chemical coupling method is toconnect two different monoclonal antibodies by chemical coupling toprepare a bispecific monoclonal antibody. For example, two differentmonoclonal antibodies chemically bind to each other, or two antibodyfragments, for example, two Fab fragments chemically bind to each other.The heterozygosis-hybridoma method is to prepare a bispecific monoclonalantibody by a cell hybridization method or a ternary hybridoma method,where the cell hybridoma or the ternary hybridoma is obtained by thefusion of constructed hybridomas or the fusion of a constructedhybridoma and lymphocytes derived from mice. Although these techniquesare used to manufacture BiAb, various generation problems make suchcomplexes difficult to use, such as the generation of mixed populationscontaining different combinations of antigen-binding sites, difficultiesin protein expression, the need for purifying the target BiAb, lowyields, and high production costs.

Recent methods utilize genetically engineered constructs that canproduce a single homogeneous product of BiAb so that there is no needfor thorough purification to remove unwanted by-products. Suchconstructs include tandem scFv, diabodies, tandem diabodies, doublevariable domain antibodies, and heterdimeric antibodies using the Ch1/Ckdomain or DNL™ motifs (Chames & Baty, Curr. Opin. Drug. Discov. Devel.,12: 276-83, 2009; Chames & Baty, mAbs, 1: 539-47). Related purificationtechniques are well known.

Tumor Surface Antigen

The term “tumor surface antigen” refers to antigens that are or can bepresent on the tumor cells or on the inner surface of the tumor cells.Some cancer cell antigens are also expressed on the surface of somenormal cells, which thus may be referred to as tumor-associatedantigens. These tumor-associated antigens may be overexpressed on tumorcells compared with their expression on normal cells, or are susceptibleto binding to antibodies in tumor cells due to the less compactstructure of the tumor tissue compared with normal tissues. Theseantigens may be presented solely by tumor cells and not by normal cells.Tumor antigens may also be expressed only on tumor cells or mayrepresent tumor-specific mutations compared with normal cells. Thecorresponding antigens may be called tumor-specific antigens.

The “tumor-associated antigen” can trigger an immune response in hostand can be used for identifying tumor cells and as a possible candidatein cancer therapy. Such an antigen may include normal proteins thatevade the immune system well, proteins that are usually produced in verysmall amounts, proteins that are usually produced only in certaindevelopmental stages, or proteins whose structure is modified bymutations.

A large number of tumor antigens are known in the art, and new tumorantigens can be readily determined through screening and identification.Non-limiting examples of tumor antigens include: α-fetoprotein (AFP),α-actinin-4, A3, antigens specific to A33 antibodies, ART-4, B7, Ba733,BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX,CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A,CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30,CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD47, CD52,CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD80,CD83, CD95, CD123, CD126, CD132, CD133, CD138, CD147, CD154, CDC27,BCMA, CS1, DLL3, DLL4, EpCAM, FLT3, gpA33, GPC-3, Her2, MEGE-A3, NYESO1,CIX, GD2, GD3, GM2, CDK-4/m, CDKN2A, CTLA-4, CXCR4, CXCR7, CXCL12,HIF-1α, Colon-specific antigen p (CSAp), CEA(CEACAM5), CEACAM6, c-Met,DAM, EGFR, EGFRvIII, EGP-1(TROP-2), EGP-2, ELF2-M, Ep-CAM, fibroblastgrowth factor (FGF), Flt-1, Flt-3, folate-binding protein, G250 antigen,GAGE, gp100, GRO-β, HLA-DR, HM1.24, human chorionic gonadotropin (HCG)and subunits thereof, HER2/neu, HMGB-1, hypoxia-inducible factor(HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IFN-λ, IL-4R,IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15,IL-17, IL-18, IL-23, IL-25, insulin-like growth factor-1 (IGF-1),KC4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR/FUT, macrophage migrationinhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3,mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13,MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, PAM4 antigen, pancreaticcancer mucoprotein, PD-1 receptor, placental growth factor, p53, PLAGL2,prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, ILGF, ILGF-1R, IL-6,IL-25, RS5, RANTES, T101, SAGE, S100, survivin, survivin-2B, TAC,TAG-72, cytotactin, TRAIL receptor, TNF-α, Tn antigen,Thomson-Friedenreich antigen, tumor necrosis antigen, VEGF, VEGFR2,VEGFR3, Cadherin, Integrin, Mesothelin, Claudin18, αVβ3, α5β1, ERBB3,IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, Mucin family, FAP, Tenascin, ED-Bfibronectin, WT-1, 17-1A-antigen, complementation factor C3, C3a, C3b,C5a, C5, angiogenesis marker, bcl-2, bcl-6, Kras, oncogene marker andoncogene products (see, for example, Sensi M et al., Clin. Cancer Res.,12: 5023-32, 2006; Parmiani J et al., J. Immunol., 178: 1975-79, 2007;Novellino L et al., Cancer Immunol. Immunother., 54: 187-207, 2005).Preferably, TAA in the present disclosure is CD19, CD20, CD22, CD30,CD38, BCMA, CS1, EpCAM, CEA, Her2, EGFR, Mucin1, CA125, GPC-3, orMesothelin.

The term also includes any variants, isotypes, derivatives, and specieshomologues of TAA that are naturally expressed by cells including tumorcells or expressed by cells transfected with TAA genes or cDNA.

TAA may be derived from any organism including, but not limited to,humans, mice, rats, rabbits or monkeys.

Target Cell and Target Cell Protein Expressed on the Target Cell

As described above, the bispecific antibody can bind to effector cellproteins and target cell proteins. For example, the target cell proteinmay be expressed on the surface of cancer cells, cells infected bypathogen, or cells mediating diseases (e.g., inflammatory and autoimmunediseases).

In some embodiments, the target cell protein can be highly expressed onthe surface of target cells, although such high-level expression is notrequired. In some embodiments, the target cell protein is not expressedor low-expressed on the surface of target cells.

When the target cell is a cancer cell, the homodimeric bispecificantibody as described herein can bind to the cancer cell antigen asdescribed above. The cancer cell antigen may be a human protein or aprotein derived from other species.

In some embodiments, the target cell protein may be a protein that isselectively expressed or overexpressed or not expressed on the surfaceof tumor cells.

In some embodiments, the target cell protein may be a protein on thesurface on cells that mediate lymphatic system-related diseases.

In other aspects, the target cell may be a cell that mediates autoimmunediseases or inflammatory diseases. For example, human eosinophils inasthma may be the target cell, and in this case, for example, theEGF-like module-containing mucin-like hormone receptor (EMR1) may be thetarget cell protein. Optionally, excess human B cells in patientssuffering from systemic lupus erythematosus may be the target cell, andin this case, for example, CD19 or CD20 may be the target cell protein.In other autoimmune diseases, excess human Th2T cells may be the targetcell, and in this case, for example, CCR4 may be the target cellprotein. Similarly, the target cell may be a fibrotic cell thatmediates, for example, atherosclerosis, chronic obstructive pulmonarydisease (COPD), liver cirrhosis, scleroderma, renal transplantationfibrosis, renal allograft nephropathy or pulmonary fibrosis (includingidiopathic pulmonary fibrosis and/or idiopathic pulmonary hypertension).For the fibrosis, for example, fibroblast activation protein α (FAPα)may be the target cell protein.

In some embodiments, the target cell protein may be a protein that isselectively expressed on the surface of infected cells. For example, inthe case of hepatitis B virus (HBV) or hepatitis C virus (HCV)infection, the target cell protein may be an envelope protein of HBV orHCV expressed on the surface of the infected cells. In otherembodiments, the target cell protein may be gp120 encoded by humanimmunodeficiency virus (HIV) on HIV-infected cells.

In some embodiments, the target cell may be a cell that mediatesinfections and infectious-related diseases.

In some embodiments, the target cell may be a cell that mediatesimmunodeficiency-related diseases.

In some embodiments, the target cell may be a cell that mediates otherrelated diseases, including diseases well known in the art or about tobe discovered in the future.

The bispecific antibody may bind to target cell proteins from species ofmice, rats, rabbits, New World monkeys, and/or Old World monkeys. Thespecies include, but are not limited to, the following species: Musmusculus, Rat tusrattus, Rattus norvegicus, Cynomolgus monkeys, Macacafascicularis, Hamadryas baboon, Papio hamadryas, Guinea baboon, Papiopapio, Olive baboon, Papio anubis, Yellow baboon, Papio cynocephalus,Chacma baboon, Papio ursinus, Callithrix jacchus, Saguinus oedipus andSaimiri sciureus.

Cancer

The term “cancer” refers to a broad class of diseases characterized byuncontrolled growth of abnormal cells in vivo. “Cancer” includes benignand malignant cancers as well as dormant tumors or micrometastases.Cancer includes primary malignant cells or tumors (e.g., tumors in whichcells have not migrated to a site other than the site of the originalmalignant disease or tumor in the subject) and secondary malignant cellsor tumors (e.g., tumors resulting from metastasis in which cells aremetastasized to malignant cells or tumor cells and then migrate to asecondary site different from the original tumor site). Cancers alsoinclude hematologic malignancies. “Hematologic malignancies” includelymphomas, leukemias, myelomas or lymphoid malignancies, as well asspleen cancer and lymph node tumors.

In a preferred embodiment, the bispecific antibody of the presentdisclosure or nucleic acids or polynucleotides encoding the antibody ofthe present disclosure or immunoconjugates or pharmaceuticalcompositions or combination therapies are useful for the treatment,prevention or alleviation of cancer. Examples of cancers include, butare not limited to, carcinomas, lymphomas, glioblastomas, melanomas,sarcomas, leukemia, myelomas or lymphoid malignancies. More specificexamples of such cancers are described below and include: squamous cellcarcinoma (e.g., epithelial squamous cell carcinoma), Ewing's sarcoma,Wilms' tumor, astrocytoma, lung cancer (including small-cell lungcancer, non-small-cell lung cancer, lung adenocarcinoma, and lungsquamous-cell carcinoma), peritoneal carcinoma, hepatocellularcarcinoma, stomach or gastric cancer (including gastrointestinalcancer), pancreatic cancer, glioblastoma multiforme, cervical cancer,ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellularcarcinoma, neuroendocrine tumor, medullary thyroid cancer,differentiated thyroid cancer, breast cancer, ovarian cancer, coloncancer, rectal cancer, endometrial or uterine carcinoma, salivary glandtumors, kidney or renal carcinoma, prostate cancer, vaginal cancer, analcancer, penile cancer, and head and neck cancer.

Other examples of cancers or malignancies include, but are not limitedto, childhood acute lymphoblastic leukemia, acute lymphoblasticleukemia, acute lymphocytic leukemia, acute myelogenous leukemia,adrenocortical carcinoma, adult (primary) hepatocellular carcinoma,adult (primary) liver cancer, adult acute lymphocytic leukemia, adultacute myelogenous leukemia, adult Hodgkin's lymphoma, adult lymphocyticlymphoma, adult non-Hodgkin's lymphoma, adult primary liver cancer,adult soft tissue sarcoma, AIDS-related lymphoma, AIDS-relatedmalignancies, anal cancer, astrocytoma, cholangiocarcinoma, bladdercancer, bone cancer, brain stem glioma, brain tumor, breast cancer,renal pelvis and ureter cancer, central nervous system (primary)lymphoma, central nervous system lymphoma, cerebellar astrocytoma,cerebral astrocytoma, cervical cancer, childhood (primary)hepatocellular carcinoma, childhood (primary) liver cancer, childhoodacute lymphoblastic leukemia, childhood acute myelogenous leukemia,childhood brain stem glioma, childhood cerebellar astrocytoma, childhoodcerebral astrocytoma, childhood extracranial blastoma, childhoodHodgkin's disease, childhood Hodgkin's lymphoma, childhood hypothalamicand visual pathway glioma, childhood lymphoblastic leukemia, childhoodmedulloblastoma, childhood non-Hodgkin's lymphoma, childhood pineal andsupratentorial primitive neuroectodermal tumors, childhood primary livercancer, childhood rhabdomyosarcoma, childhood soft-tissue sarcoma,childhood visual pathway and hypothalamic glioma, chronic lymphocyticleukemia, chronic myelogenous leukemia, colon cancer, cutaneous T-celllymphoma, endocrine pancreas islet cell carcinoma, endometrial cancer,ependymoma, epithelial cancer, esophageal cancer, Ewing's sarcoma andrelated tumors, exocrine pancreatic cancer, extracranial blastoma,extragonadal blastoma, cholangiocarcinoma, retinoblastoma, female breastcancer, Gaucher's disease, gallbladder carcinoma, gastric cancer,gastrointestinal benign tumor, gastrointestinal tumors, blastoma,gestational trophoblastic tumor, hairy cell leukemia, head and neckcancer, hepatocellular carcinoma, Hodgkin's lymphoma,hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancers,intraocular melanoma, islet cell carcinoma, islet cell pancreaticcancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oralcavity cancer, liver cancer, lung cancer, lymphoproliferative disorders,macroglobulinemia, male breast cancer, malignant mesothelioma, malignantthymoma, medulloblastoma, melanoma, mesothelioma, metastatic primarylatent squamous neck cancer, metastatic primary squamous neck cancer,metastatic squamous neck cancer, multiple myeloma, multiplemyeloma/plasma cell neoplasm, myelodysplastic syndrome, myelogenousleukemia, myeloid leukemia, myeloproliferative disorders, nasal cavityand paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma,non-Hodgkin's lymphoma, non-melanoma skin cancer, non-small-cell lungcancer, metastatic primary latent metastatic squamous neck cancer,oropharyngeal cancer, osteosarcoma/malignant fibrous sarcoma,osteosarcoma/malignant fibrous histiocytoma, osteosarcoma/malignantfibrous histiocytoma of bone, ovarian epicytoma, ovarian blastoma,ovarian low malignant potential tumor, pancreatic cancer,paraproteinemias, polycythemia vera, parathyroid cancer, penile cancer,pheochromocytoma, pituitary tumor, primary central nervous systemlymphoma, primary liver cancer, prostate cancer, rectal cancer, renalcell cancer, renal pelvis and ureter cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, sarcoidosis sarcomas, Sezarysyndrome, skin cancer, small-cell lung cancer, small intestine cancer,soft-tissue sarcoma, squamous neck cancer, stomach cancer,supratentorial primitive neuroectodermal and pineal tumors, T-celllymphoma, testicular cancer, thymoma, thyroid cancer, transitional cellcancer of the renal pelvis and ureter, transitional renal pelvis andureter cancer, trophoblastic tumors, ureter and renal pelvis cellcancer, urethral cancer, uterine cancer, uterine sarcoma, vaginalcancer, visual pathway and hypothalamic glioma, vulvar cancer,Waldenstrom's macroglobulinemia, Wilm's tumor, and any otherhyperproliferative disease, besides neoplasia, located in an organsystem listed above.

Combination Therapy

The present disclosure relates to uses of a combination of thebispecific antibody or nucleic acids or polynucleotides encoding theantibody of the present disclosure or immunoconjugates or pharmaceuticalcompositions and one or more active therapeutic agents (e.g.,chemotherapeutic agents) or other prophylactic or therapeutic modes(e.g., radiation). In such combination therapies, the various activeagents often have different complementary mechanisms of action, and thecombination therapy may lead to synergistic effects. The combinationtherapy includes therapeutic agents that affect immune responses (e.g.,an enhanced or activated response) and therapeutic agents that affect(e.g., inhibit or kill) tumor/cancer cells. The combination therapy mayreduce the likelihood of drug-resistant cancer cells. The combinationtherapy may allow the dose reduction of one or more reagents to reduceor eliminate adverse effects associated with the one or more reagents.Such combination therapies may have synergistic therapeutic orprophylactic effects on underlying diseases, disorders or symptoms.

The “combination” includes therapies that can be administeredseparately, for example, separate formulations for individualadministration (e.g., which may be provided in a kit), and therapiesthat can be administered together in a single formulation (i.e.,“co-formulation”). In some embodiments, the bispecific antibody of thepresent disclosure or nucleic acids or polynucleotides encoding theantibody of the present disclosure or immunoconjugates or pharmaceuticalcompositions may be administered sequentially. In some embodiments, thebispecific antibody of the present disclosure or nucleic acids orpolynucleotides encoding the antibody of the present disclosure orimmunoconjugates or pharmaceutical compositions may be administeredsimultaneously. The bispecific antibody or nucleic acids orpolynucleotides encoding the antibody of the present disclosure orimmunoconjugates or pharmaceutical compositions may be used in anymanner in combination with at least one other (active) agent.

Treatment with the bispecific antibody of the present disclosure may becombined with other treatments that are effective against theto-be-treated disease. Non-limiting examples of antibody combinationtherapies of the present disclosure include surgery, chemotherapy,radiation therapy, immunotherapy, gene therapy, DNA therapy, RNAtherapy, nanotherapy, viral therapy, and adjuvant therapy.

Combination therapies also include all other combination therapies knownin the art or developed in the future.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1-1 illustrates configurations of bispecific antibodies AB7K,AB7K4, AB7K5, AB7K6, AB7K7, and AB7K8 as shown in a, b, c, d, e and f,respectively.

FIG. 1-2 illustrates an expression plasmid map of bispecific antibodyAB7K7. The expression plasmid has a full length of 9293 bp and containsnine major gene fragments which are (1) an hCMV promoter, (2) targetgenes, (3) EMCV IRES, (4) mDHFR screening gene, (5) a Syndiscontinuation sequence, (6) an SV40 promoter, (7) Kalamycin resistancegene; (8) an SV40 termination sequence, and (9) a PUC replicon.

FIG. 1-3 illustrates SEC-HPLC detection results of a purified sample ofbispecific antibody AB7K7.

FIG. 1-4 illustrates SDS-PAGE electrophoresis results of a purifiedsample of bispecific antibody AB7K7.

FIG. 1-5 illustrates SDS-PAGE results of bispecific antibody AB7K7 in anacceleration test at 25° C.

FIG. 1-6 illustrates SDS-PAGE results of bispecific antibody AB7K7 in afreeze-thaw test.

FIG. 2-1 illustrates abilities, detected by FACS, of bispecificantibodies AB7K and AB7K4 to bind to tumor cells BT474.

FIG. 2-2 illustrates abilities, detected by FACS, of bispecificantibodies AB7K and AB7K5 to bind to tumor cells BT474.

FIG. 2-3 illustrates abilities, detected by FACS, of bispecificantibodies AB7K and AB7K6 to bind to tumor cells BT474.

FIG. 2-4 illustrates abilities, detected by FACS, of bispecificantibodies AB7K and AB7K7 to bind to tumor cells BT474.

FIG. 2-5 illustrates an ability, detected by FACS, of bispecificantibody AB7K8 to bind to tumor cells BT474.

FIG. 2-6 illustrates abilities, detected by FACS, of bispecificantibodies AB7K and AB7K4 to bind to effector cells CIK.

FIG. 2-7 illustrates abilities, detected by FACS, of bispecificantibodies AB7K and AB7K5 to bind to effector cells CIK.

FIG. 2-8 illustrates an ability, detected by FACS, of bispecificantibody AB7K6 to bind to effector cells CIK.

FIG. 2-9 illustrates abilities, detected by FACS, of bispecificantibodies AB7K and AB7K7 to bind to effector cells CIK.

FIG. 2-10 illustrates an ability, detected by FACS, of bispecificantibody AB7K8 to bind to effector cells CIK.

FIG. 2-11 illustrates an ability, detected by FACS, of bispecificantibody AB7K to bind to cynomolgus monkey T cells.

FIG. 2-12 illustrates abilities, detected by ELISA, of fiveAnti-Her2×CD3 bispecific antibodies to bind to CD3 molecules and Her2molecules.

FIG. 2-13 illustrates abilities, detected by a microplate reader, offive Anti-Her2×CD3 bispecific antibodies to activate Jurkat T cells of areporter gene cell strain.

FIG. 2-14 illustrates structural modeling of a CTP linker and anti-CD3scFv VH.

FIG. 2-15 illustrates structural modeling of a GS linker and anti-CD3scFv VH.

FIG. 2-16 illustrates a molecular docking model of anti-CD3 scFv and aCD3 epsilon chain.

FIG. 3-1 illustrates in vivo anti-tumor effects of bispecific antibodiesAB7K4 and AB7K7 in an NCG mouse model of transplanted tumor constructedby subcutaneously co-inoculating human CIK cells and HCC1954 cells.

FIG. 3-2 illustrates an in vivo anti-tumor effect of bispecific antibodyAB7K7 in an NPG mouse model of transplanted tumor constructed bysubcutaneously co-inoculating human CIK cells and human breast cancercells HCC1954.

FIG. 3-3 illustrates in vivo anti-tumor effects of bispecific antibodiesAB7K7 and AB7K8 at different administration frequencies in an NPG mousemodel of transplanted tumor constructed by subcutaneously co-inoculatinghuman CIK cells and human breast cancer cells HCC1954.

FIG. 3-4 illustrates an in vivo anti-tumor effect of bispecific antibodyAB7K7 in an NPG mouse of transplanted tumor constructed bysubcutaneously co-inoculating human CIK cells and SK-OV-3 cells.

FIG. 3-5 illustrates an in vivo anti-tumor effect of bispecific antibodyAB7K7 in an NPG mouse of transplanted tumor constructed bysubcutaneously co-inoculating human CIK cells and HT-29 cells.

FIG. 3-6 illustrates an in vivo anti-tumor effect of bispecific antibodyAB7K7 in a CD34 immune-reconstituted NPG mouse model of transplantedtumor constructed by subcutaneously inoculating human breast cancercells HCC1954.

FIG. 3-7 illustrates an in vivo anti-tumor effect of bispecific antibodyAB7K7 in a PBMC immune-reconstituted NPG mouse model of transplantedtumor constructed by subcutaneously inoculating human breast cancercells HCC1954.

FIG. 4-1 illustrates in vivo anti-tumor effects of bispecific antibodiesAB7K4 and AB7K7 in an NCG mouse model of transplanted tumor constructedby subcutaneously co-inoculating human CIK cells and human Burkkit'slymphoma Raji cells.

FIG. 4-2 illustrates an anti-tumor effect of bispecific antibody AB7K7in an NPG mouse model of transplanted tumor constructed bysubcutaneously inoculating human breast cancer cells HCC1954.

FIG. 4-3 illustrates changes of weights in normal cynomolgus monkeysadministered with bispecific antibodies AB7K7 and AB7K8 multiple times.

FIG. 5-1 illustrates concentration-time curves of bispecific antibodyAB7K7 in SD rats by two ELISA methods.

FIG. 5-2 illustrates concentration-time curves of bispecific antibodyAB7K8 in SD rats by two ELISA methods.

FIG. 5-3 illustrates concentration-time curves of bispecific antibodiesAB7K7 and AB7K8 in cynomolgus monkeys.

FIG. 5-4 illustrates abilities, detected at a pH of 6.0, of bispecificantibodies AB7K, AB7K5, and AB7K7 to bind to FcRn.

FIG. 5-5 illustrates abilities, detected at a pH of 7.0, of bispecificantibodies AB7K, AB7K5, and AB7K7 to bind to FcRn.

FIG. 6-1 illustrates an in vivo anti-tumor effect of bispecific antibodyAB9K in a NOD-SCID mouse of transplanted tumor model constructed bysubcutaneously co-inoculating human PBMC cells and Huh-7 cells.

FIG. 6-2 illustrates an in vivo anti-tumor effect of bispecific antibodyAB9K in a CD34 immune-reconstituted NPG mouse model of transplantedtumor constructed by subcutaneously inoculating human liver cancer cellsHuh-7.

FIG. 6-3 illustrates an in vivo anti-tumor effect of bispecific antibodyAB9K in a CD34 immune-reconstituted NPG mouse model of transplantedtumor constructed by subcutaneously inoculating human liver cancer cellsHuh-7.

FIG. 7-1 illustrates an ability, detected by flow cytometry, ofbispecific antibody AB2K to bind to CD20-positive tumor cells.

FIG. 7-2 illustrates abilities of bispecific antibodies AB2K and AB7K7to mediate effector cells to kill Raji-luc cells.

FIG. 7-3 illustrates abilities, detected by reporter gene assay, ofbispecific antibodies AB2K and AB7K7 to activate Jurkat NFATRE Luccells.

FIG. 7-4 illustrates an in vivo anti-tumor effect of bispecific antibodyAB2K in an NPG mouse model of transplanted tumor constructed bysubcutaneously co-inoculating human CIK cells and human Burkkit'slymphoma Raji cells.

FIG. 7-5 illustrates an in vivo anti-tumor effect of bispecific antibodyAB2K in an NPG mouse model of transplanted tumor constructed bysubcutaneously co-inoculating human CIK cells and human Burkkit'slymphoma Daudi cells.

FIG. 8 illustrates changes of leukocytes and lymphocytes in normalcynomolgus monkeys administered with bispecific antibody AB2K multipletimes.

FIG. 9-1 illustrates an ability, detected by FACS, of an Anti-CD19×CD3bispecific antibody to bind to tumor cells Raji.

FIG. 9-2 illustrates an ability, detected by FACS, of an Anti-CD19×CD3bispecific antibody to bind to effector cells CIK.

FIG. 9-3 illustrates abilities, detected by FACS, of bispecificantibodies AB1K2 and AB23P10 to bind to cynomolgus monkey T cells.

FIG. 9-4 illustrates abilities, detected through an ELISA, of fourAnti-CD19×CD3 bispecific antibodies to bind to CD3 molecules and CD19molecules.

FIG. 9-5 illustrates abilities, detected by a microplate reader, ofbispecific antibodies AB1K2 and AB23P8 to activate Jurkat T cells of areporter gene cell strain.

FIG. 9-6 illustrates abilities, detected through a microplate reader, offour Anti-CD19×CD3 bispecific antibodies to activate Jurkat T cells of areporter gene cell strain.

FIG. 10-1 illustrates binding of AB11K to tumor cells that overexpressantigen Mucin1 and to primary T cells of human or cynomolgus monkeys.

FIG. 10-2 illustrates an ability of AB11K to mediate expanded T cells tokill tumor cells.

FIG. 10-3 illustrates an ability of AB11K to mediate PBMC to kill tumorcells.

FIG. 10-4 illustrates an ability of AB11K to specifically activate Tcells.

FIG. 11 illustrates an in vivo anti-tumor effect of bispecific antibodyAB8K in an NPG mouse model of transplanted tumor constructed bysubcutaneously co-inoculating human CIK cells and human skin cancercells A431.

DETAILED DESCRIPTION

The present disclosure is further described through examples that shouldnot be construed as further limitations. All drawings, all referencedocuments, and the contents of patents and published patent applicationscited in the entire application are expressly incorporated herein byreference.

Example 1 Design and Preparation of Anti-Her2×CD3 Bispecific AntibodiesHaving Different Structures 1.1 Design of Bispecific Antibodies HavingDifferent Structures

In order to screen bispecific antibodies having suitable configuration,bispecific antibodies having six different configurations were designedfor Her2 and CD3, among which AB7K5, AB7K6, and AB7K8 are single-chainbivalent bispecific antibodies while AB7K, AB7K4, and AB7K7 aredouble-chain tetravalent bispecific antibodies (see FIG. 1-1), whereonly AB7K8 is free of Fc fragments. Specifically, the configuration ofthe bispecific antibodies with the above four configurations and theircomposition from the N-terminus to the C-terminus as well as their aminoacid sequence numbers are shown in Table 1-1. The specific structuralcomposition properties of the six bispecific antibodies are describedbelow:

Bispecific antibody AB7K consists of an anti-Her2 full-length antibodywhose two heavy chains are each linked at the C-terminus to an anti-CD3scFv domain by a linker peptide (L1). For the amino acid sequence of theintact antibody against Her2 contained in AB7K, reference is made to thesequence of monoclonal antibody Herceptin® (IMGT database INN 7637),wherein AB7K contains an Fc fragment from human IgG1 and has D356E/L358Mmutations (EU numbering). The linker peptide L1 consists of a flexiblepeptide and a rigid peptide, wherein the composition of the flexiblepeptide is GS(GGGGS)₃ and the rigid peptide isSSSSKAPPPSLPSPSRLPGPSDTPILPQ, wherein the composition of the linkerpeptide L2 between VH and VL of the anti-CD3 scFv is (GGGGS)₃.

Bispecific antibody AB7K4 consists of an anti-Her2 full-length antibodywhose two light chains are each linked at the C-terminus to an anti-CD3scFv domain by a linker peptide (L1). For the amino acid sequence of theheavy chain variable region of the intact antibody against Her2contained in AB7K4, reference is made to the available region sequenceof the monoclonal antibody Herceptin®, and for the light chain aminoacid sequence thereof, reference is made to the light chain amino acidsequence of the monoclonal antibody Herceptin® (IMGT database INN 7637).The AB7K4 heavy chain contains an Fc fragment from human IgG1, hasmultiple amino acid substitutions/replacements, which are L234A, L235A,T250Q, N297A, P331S, and M428L (EU numbering), respectively, and alsohas a deleted/missed K447 (EU numbering) at the C-terminus of the Fcfragment. The linker peptide L1 consists of a flexible peptide and arigid peptide, wherein the composition of the flexible peptide isG₂(GGGGS)₃ and the rigid peptide is SSSSKAPPPS, wherein the compositionof the linker peptide L2 between VH and VL of the anti-CD3 scFv is(GGGGS)₃.

Bispecific antibody AB7K5 consists of an anti-Her2 scFv, an Fc fragment,a linker peptide L2 and an anti-CD3 scFv, which are sequentiallyconnected in series, wherein VH and VL in the anti-Her2 scFv areconnected by a linker peptide L1, and VH and VL in the anti-CD3 scFv areconnected by a linker peptide L3. For the amino acid sequence of thescFv against Her2 contained in AB7K5, reference is made to the availableregion sequence of the monoclonal antibody Herceptin®. The AB7K5contains an Fc fragment from human IgG1 and has multiple amino acidsubstitutions/replacements, which are C226S, C229S, L234A, L235A, T250Q,N297A, P331S, T366R, L368H, K409T, and M428L (EU numbering),respectively. Mutations at the five sites C226S, C229S, T366R, L368H,and K409T can prevent polymerization between Fc fragments, therebypromoting the formation of a single-chain bivalent bispecific antibody.ADCC and CDC activities are removed from Fc fragments carrying themutations L234A/L235A/P331S. The mutations T250Q/M428L can enhance thebinding affinity of Fc fragments for the receptor FcRn, therebyextending the half-life. The mutation N297A avoids antibodyglycosylation and loses the ability to bind FcγRs. In addition, K447 (EUnumbering) at the C-terminus of the Fc fragment is deleted/missed,thereby eliminating the charge heterogeneity of the antibody. The linkerpeptide (L2) consists of a flexible peptide and a rigid peptide, whereinthe flexible peptide is G₂(GGGGS)₃ and the rigid peptide is SSSSKAPPPS.The composition of the linker peptides L1 and L3 inside each scFv is(GGGGS)₃.

Bispecific antibody AB7K6 consists of an anti-Her2 scFv, a linkerpeptide L2, an anti-CD3 scFv, and an Fc fragment, which are sequentiallyconnected in series, wherein VH and VL in the anti-Her2 scFv areconnected by a linker peptide L1, and VH and VL in the anti-CD3 scFv areconnected by a linker peptide L3. The AB7K6 contains an Fc fragment fromhuman IgG1 and has multiple amino acid substitutions/replacements, whichare C226S, C229S, L234A, L235A, T250Q, N297A, P331S, T366R, L368H,K409T, and M428L (EU numbering), respectively. Mutations at the fivesites C226S, C229S, T366R, L368H, and K409T can prevent polymerizationbetween Fc fragments, thereby promoting the formation of a single-chainbivalent bispecific antibody. ADCC and CDC activities are removed fromFc fragments carrying the mutations L234A/L235A/P331S. The mutationsT250Q/M428L can enhance the binding affinity of Fc fragments for thereceptor FcRn, thereby extending the half-life. The mutation N297Aavoids antibody glycosylation and loses the ability to bind FcγRs. Inaddition, K447 (EU numbering) at the C-terminus of the Fc fragment isdeleted/missed, thereby eliminating the charge heterogeneity of theantibody. The linker peptide (L2) consists of a flexible peptide and arigid peptide, wherein the flexible peptide is G₂(GGGGS)₃ and the rigidpeptide is SSSSKAPPPS. The composition of the linker peptides L1 and L3inside each scFv is (GGGGS)₃.

Bispecific antibody AB7K7 consists of an anti-Her2 scFv, a linkerpeptide L2, an anti-CD3 scFv, and an Fc fragment, which are sequentiallyconnected in series, wherein VH and VL in the anti-Her2 scFv areconnected by a linker peptide L1, and VH and VL in the anti-CD3 scFv areconnected by a linker peptide L3. For the amino acid sequence of thescFv against Her2 contained in AB7K7, reference is made to the availableregion sequence of the monoclonal antibody Herceptin®. The AB7K7contains an Fc fragment from human IgG1, and has multiple amino acidsubstitutions/replacements, which were L234A, L235A, T250Q, N297A,P331S, and M428L (EU numbering), respectively, and also has adeleted/missed K447 (EU numbering) at the C-terminus of the Fc fragment.The linker peptide (L2) consists of a flexible peptide and a rigidpeptide, wherein the flexible peptide is G₂(GGGGS)₃ and the rigidpeptide is SSSSKAPPPS. The composition of the linker peptides L1 and L3inside each scFv is (GGGGS)₃.

Bispecific antibody AB7K8 consists of an anti-Her2 scFv, a linkerpeptide L2, an anti-CD3 scFv, and a His-tag, which are sequentiallyconnected in series, wherein VH and VL in the anti-Her2 scFv areconnected by a linker peptide L1, and VH and VL in the anti-CD3 scFv areconnected by a linker peptide L3. For the amino acid sequence of thescFv against Her2 contained in AB7K8, reference is made to the availableregion sequence of the monoclonal antibody Herceptin®. AB7K8 is addedwith a His-tag at the C-terminus of the anti-CD3 scFv to facilitateantibody purification, wherein the composition of the His tag isHHHHHHHH. The linker peptide (L2) consists of a flexible peptide and arigid peptide, wherein the flexible peptide is G₂(GGGGS)₃ and the rigidpeptide is SSSSKAPPPS. The composition of the linker peptides L1 and L3inside each scFv is (GGGGS)₃.

VH and VL amino acid sequences of the anti-CD3 scFv contained in theabove six bispecific antibodies are as shown in SEQ ID NO: 247 and SEQID NO: 248, respectively, wherein VH and VL are connected to each otherby (GGGGS)₃. The monoclonal antibody (designated as CD3-3) specificallybinds to human and cynomolgus monkey CD3 antigens and has a weak bindingaffinity for CD3.

TABLE 1-1 Bispecific antibodies with four different structures againstHer2 and CD3 Amino acid Code Configuration Composition from N-terminusto C-terminus sequence No. Single-chain AB7K5 scFv-mFc-scFv[VH-L1-VL]_(Her2)-mFc-L2-[VH-L3-VL]_(CD3) SEQ ID NO: 1 bivalent AB7K6scFv-scFv-mFc [VH-L1-VL]_(Her2) L2-[VH-L3-VL]_(CD3)-mFc SEQ ID NO: 2bispecific AB7K8 scFv-scFv-His tag[VH-L1-VL]_(Her2)-L2-[VH-L3-VL]_(CD3)-H₈ SEQ ID NO: antibody 3Double-chain AB7K IgG(H)-scFv [VH-CH]_(Her2)-L1-[VH-L2-VL]_(CD3) SEQ IDNO: 4 tetravalent [VL-CL]_(Her2) SEQ ID NO: 5 bispecific AB7K4IgG(L)-scFv [VH-CH]_(Her2) SEQ ID NO: 6 antibody[VL-CL]_(Her2)-L1-[VH-L2-VL]_(CD3) SEQ ID NO: 7 AB7K7 scFv-scFv-mFc[VH-L1-VL]_(Her2)-L2-[VH-L3-VL]_(CD3)-mFc SEQ ID NO: 8 Note: Ln in thetable represents the linker peptides between different structural units,wherein n is numbered sequentially in the order of the linker peptidescontained between different structural units from the N-terminus to theC-terminus of the bispecific antibody.

1.2 Construction of an Expression Vector of a Bispecific AntibodyMolecule

Genes encoding the preceding five bispecific antibodies were synthesizedby conventional molecular biology method, and cDNAs encoding theobtained fusion genes were inserted into corresponding restrictionendonuclease sites of eukaryotic expression plasmids pCMAB2M modifiedwith PCDNA3.1. The heavy chains and light chains of AB7K and AB7K4 maybe constructed into one vector or separately into two different vectors.For example, the expression plasmid map of AB7K7 is as shown in FIG.1-2, wherein the plasmid contains cytomegalovirus early promoter. Thepromoter is an enhancer required for the high-level expression offoreign genes in mammalian cells. The plasmid pCMAB2M also contains aselective marker so that kanamycin resistance may be present in bacteriaand G418 resistance may be present in mammalian cells. In addition, whenhost cells are deficient in the expression of DHFR genes, the pCMAB2Mexpression vector contains mouse dihydrofolate reductase (DHFR) genes sothat target genes and the DHFR genes can be co-amplified in the presenceof methotrexate (MTX) (see U.S. Pat. No. 4,399,216).

1.3 Expression of Bispecific Antibody Molecules

The preceding constructed expression plasmids were transfected into amammalian host cell line to express bispecific antibodies. To maintainstable and high-level expression, the preferred host cell line is a DHFRdeficient CHO-cell (see U.S. Pat. No. 4,818,679), and in this Example,the host cell was selected as the CHO-derived cell strain DXB11. Apreferred transfection method is electroporation. Other methods,including calcium phosphate co-precipitation and lipofection may also beused. During electroporation, 50 μg of expression vector plasmids DNAwere added to 5×10⁷ cells in a cuvette with a Gene Pulser Electroporator(Bio-Rad Laboratories, Hercules, Calif.) with an electric field of 300 Vand capacitance of 1500 μFd. After two-day transfection, the medium waschanged to a growth medium containing 0.6 mg/mL G418. Transfectants weresubcloned by the limiting dilution method, and the secretion rate ofeach cell line was determined by ELISA. Cell strains expressingbispecific antibodies at high levels were screened.

To achieve the high-level expression of fusion proteins, DHFR genesinhibited by MTX should be used for co-amplification. The transfectedfusion protein genes were co-amplified with the DHFR genes in growthmedia containing MTX with increasing concentrations. Subclones that werepositive for DHFR expression were subjected to limiting dilution withgradually increased pressure to screen transfectants capable of growingin media with MTX of up to 6 μM. The secretion rates of thetransfectants were determined and cell lines with high foreign proteinexpression were screened. Cell lines with a secretion rate of greaterthan about 5 μg/10⁶ (millions) cells/24 hours (preferably about 15μg/10⁶ cells/24 hours) were adaptively suspended using a serum-freemedium. Cell supernatants were collected and bispecific antibodies wereseparated and purified.

Hereinafter, the purification process, stability, in vitro and in vivobiological functions, safety, and pharmacokinetics of the bispecificantibodies of several configurations were evaluated to screen thebispecific antibody of an appropriate configuration.

1.4 Purification Process and Stability Detection of BispecificAntibodies

Antibodies are generally purified by a three-step purification strategy:crude purification (sample capture), intermediate purification, and finepurification. In the crude purification stage, the target antibodies aregenerally captured by affinity chromatography which can effectivelyremove a large number of impurities such as heterologous proteins,nucleic acids, endotoxins, and viruses from the sample. The intermediatepurification is often carried out using hydrophobic chromatography orCHT hydroxyapatite chromatography to remove most of the remainingimpurity proteins and polymers. Fine purification is mostly carried outusing anion exchange chromatography or gel filtration chromatography(molecular sieve) to remove the small or trace amount of remainingimpurity proteins whose nature is similar to the nature of the targetantibodies and further to remove contaminants such as HCP and DNA.

In the present disclosure, the culture supernatant of bispecificantibody AB7K8 fused with His-tag can be crudely purified using a metalchelation affinity chromatography column (e.g., HisTrap FF from GE). Thebispecific antibodies AB7K4, AB7K5, AB7K6, AB7K, and AB7K7 containing Fccan be crudely purified using a Protein A/G affinity chromatographycolumn (e.g., Mabselect SURE from GE). The products obtained after theabove crude purification are then subjected to the intermediatepurification and the fine purification to finally obtain purified targetantibodies of high purity and high quality. The preservation buffers forthe above bispecific antibodies are then replaced with PBS or othersuitable buffers using desalination columns (e.g., HiTrap desalting fromGE).

a) Purification of Double-Chain Tetravalent Bispecific Antibody AB7K7

Specific purification steps and solutions for such bispecific antibodiesof a tetravalent homodimer configuration are illustrated below by usingan example of AB7K7.

The bispecific antibody AB7K7 was purified by three-step chromatography.The three-step chromatography included affinity chromatography,hydrophobic chromatography, and anion exchange chromatography. (Theprotein purifier used in this example was AKTA pure 25 M from GE, U.S.Reagents used in this example were purchased from Sinopharm ChemicalReagent Co., Ltd and had purity at an analytical grade).

In a first step, affinity chromatography was performed. Sample captureand concentration and the removal of partial pollutants were performedusing an affinity chromatography medium MabSelect Sure from GE or othercommercially available affinity media (e.g., Diamond Protein A fromBestchrom). First, chromatography columns were equilibrated with 3-5column volumes (CVs) of an equilibration buffer (20 mM PB, 140 mM NaCl,pH 7.4) at a linear flow rate of 100-200 cm/h. The clarifiedfermentation broth was loaded at a linear flow rate of 100-200 cm/h witha load not higher than 20 mg/mL. After loading, the chromatographycolumns were equilibrated with 3-5 column volumes (CVs) of anequilibration buffer (20 mM PB, 140 mM NaCl, pH 7.4) at a linear flowrate of 100-200 cm/h to remove unbound components. The chromatographycolumns were rinsed with 3-5 column volumes of decontamination buffer 1(50 mM NaAc-HAc, 1 M NaCl, pH 5.0) at a linear flow rate of 100-200 cm/hto remove partial pollutants. The chromatography columns wereequilibrated with 3-5 column volumes (CVs) of decontamination buffer 2(50 mM NaAc-HAc, pH 5.0) at a linear flow rate of 100-200 cm/h. Thetarget product was eluted using an elution buffer (40 mM NaAc-HAc, pH3.5) at a linear flow rate not higher than 100 cm/h and target peakswere collected.

In a second step, hydrophobic chromatography was performed. Intermediatepurification was performed using Butyl HP from Bestchrom or othercommercially available hydrophobic chromatography media to reduce thecontent of polymers. After the target proteins were polymerized, sincethe polymers and monomers differed in property such as chargecharacteristics and hydrophobicity, the polymers and the monomers couldbe separated on the basis of the above differences between them. First,chromatography columns were equilibrated with 3-5 column volumes (CVs)of an equilibration buffer (20 mM PB, 0.3 M (NH₄)₂SO₄, pH 7.0) at alinear flow rate of 100-200 cm/h. The target proteins separated throughthe affinity chromatography in the first step were subjected toconductivity adjustment to 40-50 ms/cm with the solution of 2 M(NH₄)₂SO₄ and then loaded with a load controlled to be less than 20mg/mL. After loading, the chromatography columns were rinsed with 3-5column volumes (CVs) of an equilibration buffer (20 mM PB, 0.3 M(NH₄)₂SO₄, pH 7.0) at a linear flow rate of 100-200 cm/h. Finally, thetarget proteins were eluted using 3-5 column volumes (CVs) of an elutionbuffer (20 mM PB, pH 7.0) with gradients of 40%, 80% and 100% at alinear flow rate not higher than 100 cm/h. Eluted fractions werecollected and sent for SEC-HPLC, respectively. Target components withthe percentage of monomers being greater than 90% were combined forchromatography in the next step.

In a third step, anion exchange chromatography was performed. Finepurification was performed by using Q-HP from Bestchrom or othercommercially available anion exchange chromatography media (e.g., Q HPfrom GE, Toyopearl GigaCap Q-650 from TOSOH, DEAE Beads 6FF fromSmart-Lifesciences, Generik MC-Q from Sepax Technologies, Inc, FractogelEMD TMAE from Merck, and Q Ceramic HyperD F from Pall) to separatestructural variants and further remove pollutants such as HCP and DNA.First, chromatography columns were rinsed with 3-5 column volumes (CVs)of an equilibration buffer (20 mM PB, pH 7.0) at a linear flow rate of100-200 cm/h. The target proteins separated through the hydroxyapatitechromatography in the second step were loaded and through-flow peakswere collected. After loading, the chromatography columns were rinsedwith 3-5 column volumes (CVs) of an equilibration buffer (20 mM PB, pH7.0) at a linear flow rate of 100-200 cm/h. The through-flow componentswere collected and sent for the detection of protein content, SEC-HPLCand electrophoresis.

The SEC-HPLC purity results and SDS-PAGE electrophoresis results of thesamples are shown in FIG. 1-3 and FIG. 1-4. The SEC-HPLC results showthat the purity of the main peak of the bispecific antibody was morethan 95% after three-step chromatography. The band pattern in theSDS-PAGE electrophoresis was as expected, wherein a band was shown at180 KDa in the non-reducing electrophoresis and a clear single-chainband (90 KDa) was obtained after reduction.

b) Purification of Single-Chain Bivalent Bispecific Antibodies AB7K5 andAB7K6

The bispecific antibody AB7K5 was purified by Protein A affinitychromatography and hydroxyapatite (CHT) chromatography. After theSEC-HPLC test, it was found that the purity of bispecific antibody AB7K5was low, its yield was not high, and there was a problem of extremelylow expression yield.

For another single-chain bivalent bispecific antibody AB7K6, there alsowas a problem of high process development difficulty. The bispecificantibody AB7K6 was subjected to two-step purification, that is, ProteinA affinity chromatography and molecular sieve chromatography Superdex200. After the SEC-HPLC test, it was found that it was difficult toquantify the purity of bispecific antibody AB7K6, and there was asignificant “shoulder peak” in the main peak; in addition, theexpression yield of AB7K6 was very low and very unstable. After 24 hoursof standing in a refrigerator at 4° C., it was found that the peak shapein the SEC-HPLC result was changed from two peaks to one main peak,which attributed, presumably, to the conversion from the single-chainstructure to the double-chain structure in AB7K6. From the above, it canbe seen that the current process development difficulty of AB7K6 is toohigh to achieve process scale-up and industrialization.

In summary, AB7K7 had significant advantages over AB7K5 and AB7K6 interms of process development and had advantages such as high yield,simple and efficient purification methods, and stable downstreamprocesses. The physicochemical stability of AB7K7 in different buffersystems and at different storage conditions was further studied.

c) Assay on Stability of Bispecific Antibody AB7K7

The stability of AB7K7 proteins in a citrate buffer system (20 mMcitrate, pH 5.5) and a histidine buffer system (20 mM histidine, pH 5.5)was studied, respectively. AB7K7 proteins were stored for four weeksunder accelerated conditions at 25° C. for the evaluation of proteinstability.

AB7K7 proteins were transferred to the preceding citrate buffer system(F2) and the histidine buffer system (F3), respectively, with theconcentration adjusted to 0.5 mg/mL, wherein 8% sucrose (w/v) and 0.02%PS80 (w/v) were added to both buffer systems as excipients. The abovebuffer systems were filtered using a 0.22 m PES membrane needle filter,and then vialed into 2 mL penicillin bottles, respectively, 0.8 mL ineach bottle. After the vialing, a stopper was immediately pressed andcapped. Samples were placed in different stability chambers according tothe schemes in Table 1-2. Samples were taken at each sampling point fordetection and analysis, wherein the detection terms included appearance,concentration, purity (detected by SEC-HPLC), HMW %, LMW %, andturbidity (A340) of the sample.

TABLE 1-2 Stability detection scheme Condition T₀ Sampling point anddetection term 40° C. X, Y 1 Week (W) 2 W 4 W X, Y X X, Y 25° C. 1 W 2 W4 W X, Y X X, Y Freeze-thaw 3 cycles (−70° C./ X, Y room temperature)Note: X = appearance, concentration, SEC-HPLC, SDS-PAGE (reducing &non-reducing); Y = turbidity (A340)

The appearance, concentration, turbidity and SEC-HPLC detection resultsof two preparations stored for 0-4 weeks at 25° C. are shown in Table1-3 and Table 1-4, and SDS-PAGE (reducing/non-reducing) results thereofare shown in FIG. 1-5. There was no significant change in the appearanceand concentration of the two preparations. In the SEC-HPLC results, theSEC results of the F2 and F3 preparations did not show any significantchange. The purity after 4 weeks was 97.9% and 98.2%, respectively.SDS-PAGE (reducing/non-reducing) results were generally consistent withthe trend of LMW % results, and F2 and F3 slightly changed.

To know the unfolding temperature of AB7K7 proteins in the two buffersystems, the Tm (unfolding temperature) and Tmonset (the temperature atwhich the protein begins to unfold) in the two preparations weremeasured by DSF and the results are shown in Table 1-5. Bothpreparations had low Tmonset values, and F2 and F3 had Tmonset valuesless than 45° C.

TABLE 1-3 Appearance, concentration, and turbidity results in theacceleration test at 25° C. Appearance Concentration Turbidity A340 T 01 W 2 W 4 W T 0 1 W 2 W 4 W T 0* 1 W 4 W F2 Colorless clear liquidwithout 0.46 0.46 0.45 0.46 0.003 0.004 0.002 F3 visible foreign matter0.47 0.46 0.45 0.47 0.004 0.003 0.005 *T 0 turbidity: the sample to bedetected was a sample subjected to 1 cycle of freeze-thaw.

TABLE 1-4 SEC-HPLC results in the acceleration test at 25° C. SEC-Purity% SEC-HMW % SEC-LMW % T 0 1 W 2 W 4 W T 0 1 W 2 W 4 W T 0 1 W 2 W 4 W F297.5 98.3 98.5 97.9 2.5 1.5 1.5 1.5 0 0.3 0.0 0.6 F3 97.7 98.8 98.7 98.22.3 1.2 1.3 1.2 0 0.0 0.0 0.6

TABLE 1-5 DSF results Tmonset (° C.) Tm1 (° C.) Tm2 (° C.) F2 42.0 46.060.5 F3 41.0 45.0 58.0

The stability of AB7K7 proteins in the above two buffer systems duringfreeze−thaw (−70° C./room temperature, 3 cycles of freeze-thaw) wasstudied by performing 3 cycles of freeze-thaw. The preparation anddetection solution of the sample were the same as those described above.

The appearance, concentration, turbidity and SEC-HPLC detection resultsof samples are shown in Table 1-6, and SDS-PAGE (reducing/non-reducing)results thereof are shown in FIG. 1-6. In the SDS-PAGE (non-reducing)results, there were no significant changes in the results of eachdetection item of both F2 and F3 preparations subjected to three cyclesof freeze-thaw.

TABLE 1-6 Appearance, concentration, turbidity, and SEC-HPLC results inthe freeze-thaw test Appearance Concentration Turbidity A340 SEC-Purity% SEC-HMW % SEC-LMW % T 0 FT-3 C. T 0 FT-3 C. T 0 FT-3 C. T 0 FT-3 C. T0 FT-3 C. T 0 FT-3 C. F2 Colorless Colorless 0.46 0.46 0.003 0.005 97.598.4 2.5 1.6 0 0.0 F3 clear liquid clear liquid 0.47 0.48 0.004 0.00597.7 98.5 2.3 1.4 0 0.2 without visible without visible foreign matterforeign matter *T 0 turbidity: the sample to be detected was a samplesubjected to 1 cycle of freeze-thaw.

Example 2 Evaluation of In Vitro Biological Functions of Anti-Her2×CD3Bispecific Antibodies 2.1 Detection of Binding Activities of BispecificAntibodies to Effector Cells and Target Cells (FACS) a) Detection ofBinding Activities of Bispecific Antibodies to Her2-Positive Tumor CellsBT-474 by Flow Cytometry

Tumor cells BT-474 that were positive for Her2 expression (from the cellbank of Chinese Academy of Sciences, Shanghai) were cultured, thendigested with 0.25% trypsin, and centrifuged to collect cells. Thecollected cells were resuspended with 1% PBSB, placed in 96-well platesafter the cell density was adjusted to (2×10⁶) cells/ml, 100 μl (2×10⁵cells) per well, and blocked for 0.5 hours at 4° C. The blocked cellswere centrifuged to discard the supernatant, and a series of dilutedbispecific antibodies were added to the cells. The cells were incubatedfor 1 hour at 4° C., then centrifuged to discard the supernatant, andwashed three times using a PBS solution with 1% BSA (PBSB). DilutedAF488-labeled goat anti-human IgG antibodies or murine anti-6×His IgGantibodies were added to the cells, and the cells were incubated for 1hour at 4° C. in the dark. The obtained cells were centrifuged todiscard the supernatant, and washed twice with 1% PBSB, and cells ineach well were resuspended with 100 μl of 1% paraformaldehyde (PF). Thesignal intensity was detected by flow cytometry. The analysis wasperformed with the average fluorescence intensity as the Y-axis and theantibody concentration as the X-axis through software GraphPad tocalculate the EC₅₀ value for the binding of bispecific antibodies totumor cells BT-474.

The results show that bispecific antibodies with different structureshad good binding activity to tumor cells overexpressing Her2. FIG. 2-1to FIG. 2-5 show binding curves of bispecific antibodies with differentstructures to tumor cells BT-474. As shown in Table 2-1, the EC₅₀ forthe binding of AB7K to tumor cells and the EC₅₀ for the binding of AB7K4to tumor cells both were around 5 nM, the EC₅₀ for the binding of AB7K7to tumor cells was close to 50 nM, the EC₅₀ for the binding of AB7K5 totumor cells and the EC₅₀ for the binding of AB7K8 to tumor cells bothwere greater than 100 nM, and the EC₅₀ for the binding of AB7K6 to tumorcells was greater than 200 nM.

TABLE 2-1 Detection of abilities of Anti-Her2 × CD3 bispecificantibodies to bind to tumor cells BT474 AB7K AB7K4 AB7K5 AB7K6 AB7K7AB7K8 EC₅₀ (nM) 5.009 4.388 125.0 239.9 51.98 125.3

b) Detection of Binding Activities of Bispecific Antibodies to Human TCells by FACS

PBMCs were prepared from fresh human blood by density gradientcentrifugation. The prepared PBMCs were resuspended in a 1640 mediumcontaining 10% heat-inactivated FBS, added with 2 μg/ml OKT3 foractivation for 24 h, then added with 250 IU/ml IL-2 for amplificationfor 7 days, to prepare cytokine-induced killer (CIK) cells which weredetected by flow cytometry to be positive for CD3 expression on thesurface. The to-be-detected samples were prepared and detected in thesame manner as in a) of Example 2.1. Cells resuspended with 1% PF weredetected on a machine and, with the average fluorescence intensity,analyzed by software OriginPro 8 to calculate the EC₅₀ value for thebinding of each bispecific antibody to human CIK cells.

The results show that there were great differences among the binding ofeach bispecific antibody to CIK cells (FIG. 2-6 to FIG. 2-10). As shownin Table 2-2, the EC₅₀ of AB7K was about 20 nM, which was roughly equalto the EC₅₀ of AB7K4, the EC₅₀ of AB7K7 was more than 6 times higherthan the EC₅₀ of AB7K, and the EC₅₀ of AB7K5, AB7K6 and AB7K8 was morethan 10 times higher than the EC₅₀ of AB7K.

TABLE 2-2 Detection of abilities of Anti-Her2 × CD3 bispecificantibodies to bind to effector cells CIK AB7K AB7K4 AB7K5 AB7K6 AB7K7AB7K8 EC₅₀ (nM) 20.51 19.44 375.2 241.7 132.3 504.1c) Detection of Cross-Reactivity of Bispecific Antibodies withCynomolgus Monkey CIK Cell Membrane CD3 by FACS

PBMCs were prepared from fresh cynomolgus monkey blood by densitygradient centrifugation. The prepared PBMCs were resuspended in a 1640medium containing 10% heat-inactivated FBS, added with 2 μg/ml OKT3 foractivation for 24 h, then added with 250 IU/ml IL-2 for amplificationfor 7 days, to prepare cynomolgus monkey CIK cells for use. Human CIKcells and cynomolgus monkey CIK cells were collected by centrifugation,followed by the same test procedure as in the above examples. Cellsresuspended with 1% paraformaldehyde solution were detected on a machineand, with the average fluorescence intensity, analyzed by softwareOriginPro 8 to calculate the EC₅₀ values for the binding of bispecificantibodies to human CIK cells and the EC₅₀ values for the binding ofbispecific antibodies to cynomolgus monkey CIK cells.

As shown in FIG. 2-11, the bispecific antibody AB7K bound well tocynomolgus monkey T cells, the ability of AB7K to bind to cynomolgusmonkey T cells was roughly equal to the ability of AB7K to bind to humanT cells, and the EC₅₀ for the binding of AB7K to cynomolgus monkey Tcells was approximately 26 nM as detected by flow cytometry. Bispecificantibodies AB7K4, AB7K5, AB7K6, AB7K7, and AB7K8 bound specifically tocynomolgus monkey T cells, as did AB7K.

2.2 Detection of Abilities of Bispecific Antibodies to Bind to Antigens

The binding of bispecific antibodies to soluble CD3 and Her2 wasdetected by double antigen sandwich ELISA.

Her2 proteins (SinoBiological, Beijing, Cat. No. 10004-H08H4) werediluted with PBS to a concentration of 0.1 μg/ml and added to 96-wellplates, 100 μl per well. The plates were coated at 4° C. overnight. Theplates were then blocked with 1% skimmed milk powder for 1 hour at roomtemperature. Each bispecific antibody was diluted simultaneously with a4-fold gradient for a total of 11 concentration gradients. The 96-wellplates were then washed with PBST, and then the diluted bispecificantibodies were added. Control wells without antibodies were set.Incubated for 1 hour at room temperature. Unbound bispecific antibodieswere washed away with PBST. Biotinylated CD3E and CD3D (ACRO Biosystem,Cat. No. CDD-H82W1) were mixed at 50 ng/ml with streptavdin HRP (BD,Cat. No. 554066), added in the 96-well plates, 100 μl per well, andincubated for 1 hour at room temperature. 96-well plates were washedwith PBST, and TMB was added to the plates, 100 μl per wellss. Colordevelopment was performed at room temperature for 15 minutes, and then0.2 M H₂SO₄ was added to stop the color development reaction. The lightabsorbance values at A450-620 nm were measured by a microplate reader.Analysis was performed by software OriginPro 8, and the EC₅₀ values forthe binding of bispecific antibodies to two antigens were calculated.

The results show that each bispecific antibody bound specifically toboth CD3 and Her2 molecules and exhibited good dose-dependence as theconcentration of the antibodies changed (FIG. 2-12). The abilities ofseveral bispecific antibodies to bind to soluble CD3 and Her2 are shownin Table 2-3, with EC₅₀ values ranging from 0.03 nM to 3.8 nM whichdiffer by two orders of magnitude. AB7K had the best binding activity,binding activities of AB7K4 and AB7K7 differed by one order ofmagnitude, and AB7K5 and AB7K8 had the weakest binding activity.

TABLE 2-3 Detection of abilities of Anti-Her2 × CD3 bispecificantibodies to bind to CD3 and Her2 molecules AB7K AB7K4 AB7K5 AB7K7AB7K8 EC₅₀ (nM) 0.03128 0.1518 1.004 0.1398 3.815

2.3 Evaluation of Abilities of Bispecific Antibodies to Activate T CellsThrough Reporter Gene Cell Strains

Jurkat T cells containing NFAT RE reporter genes (BPS Bioscience, Cat.No. 60621) can overexpress luciferase in the presence of bispecificantibodies and target cells, and the degree of activation of the JurkatT cells can be quantified by detecting the activity of the luciferase. Afour-parameter curve was fitted using the concentration of bispecificantibodies as the X-axis and the fluorescein signal as the Y-axis.

The test results from FIG. 2-13 show that the monoclonal antibodyHerceptin targeting Her2 cannot activate Jurkat T cells. T cells can beactivated only in the presence of both antibodies. The ability of eachantibody to activate Jurkat T cells is shown in Table 2-4. AB7K4 had thestrongest ability to activate T cells, AB7K8 had the weakest ability toactivate T cells, and their EC₅₀ values differed by one order ofmagnitude.

TABLE 2-4 Detection of abilities of Anti-Her2 × CD3 bispecificantibodies to a reporter gene cell strain that are Jurkat T cells AB7KAB7K4 AB7K5 AB7K7 AB7K8 Herceptin EC₅₀ (nM) 0.02263 0.01338 0.053570.08952 0.1575 0.009907

2.4 Abilities of Bispecific Antibodies to Mediate T Cells to Kill TumorCells

Normally cultured tumor cell lines, including SK-BR-3, MCF-7, HCC1937,NCI-N87, HCC1954 cells (all purchased from the cell bank of ChineseAcademy of Sciences, Shanghai), as target cells, were digested with0.25% trypsin to prepare single-cell suspensions, added to 96-well cellculture plates after the cell density was adjusted to 2×10⁵ cells/ml,100 μl per well, and cultured overnight. The antibodies were dilutedaccording to the test design, and added to the cells, 50 μl per well,while wells without the addition of antibodies were supplemented withthe same volume of the medium. Effector cells (human PBMCs or expandedCIK cells) whose number was five times larger than the number of targetcells, were then added, 100 μl per well. Control wells were set, andwells without the addition of effector cells were supplemented with thesame volume of the medium. After incubation for 48 hours, thesupernatant was discarded from the 96-well plates. The cells were thenwashed three times with PBS, and a complete medium containing 10% CCK-8was added, 100 μl per well, and the cells were incubated for 4 hours at37° C. The light absorbance values at A450-620 nm were measured by amicroplate reader. Analysis was performed by software OriginPro 8, andthe ability of each bispecific antibody to mediate the killing of tumorcells and the ability of the same target monoclonal antibody Herceptinto mediate the killing of tumor cells were calculated and compared.

The EC₅₀ values of each bispecific antibody to mediate effector cells tokill tumor cells are shown in Table 2-5. The results show that eachbispecific antibody exhibited a very significant killing effect on tumorcells (e.g., SK-BR-3, NCI-N87, and HCC1954) with high expression of Her2in a dose-dependent manner. Each bispecific antibody, in particularAB7K7, also exhibited a good killing effect on breast cancer cells MCF-7with low expression of Her2. Each bispecific antibody also had a goodkilling effect on the Herceptin-resistant cell strain HCC1954 while eachbispecific antibody exhibited the killing effect on the cell strainHCC1937 that was negative for Her2 expression (with little expression)only at two highest concentrations.

TABLE 2-5 EC₅₀ values of bispecific antibodies to mediate PBMCs to killdifferent tumor cells EC₅₀ (nM) AB7K7 AB7K8 AB7K5 Herceptin SK-BR-3~0.001 ~0.002 ~0.001  — ~0.001 0.011 — 0.067 MCF-7 ~0.005 0.079 0.055 —HCC1937 0.659 ~2.269 1.223 — 0.579 4.011 — >6.667  NCI-N87 0.015 0.034 —0.129 HCC1954 0.002 0.018 — 0.050 Note: ~means approximately equal to,and — means that no detection is performed.2.5 Evaluation of the Effect of GS-CTP Linker Peptide on the Ability ofAnti-CD3 scFv to Bind to CD3 Molecules by Computer Techniques

The anti-CD3 scFv VH containing the GS-CTP linker peptide wasstructurally modeled using computer software and the spatialconformation of molecular docking of anti-CD3 scFv and its antigen CD3epsilon chain was simulated and predicted.

The sequence of the GS-CTP linker peptide between anti-Her2 scFv andanti-CD3 scFv in the bispecific antibody AB7K7 is(GGGGGGSGGGGSGGGGSSSSSKAPPPS), wherein the first half of the sequence isa GS-flexible peptide (GGGGGGSGGGGSGGGGS), and the second half isCTP-rigid peptide (SSSSKAPPPS). The rigid CTP portion (SSSSKAPPPS) isconnected to the N-terminus of the anti-CD3 scFv VH. Throughthree-dimensional structural modeling using software phyre2, it is foundthat the CTP peptide fragment structurally overlays on the CDR1 regionof VH of the anti-CD3 scFv (FIG. 2-14), which may hinder or disrupt thebinding of the CD3 antibody to its antigen. The VH of the anti-CD3 scFvconnected to the GS linker peptide (containing only the GS flexiblepeptide with the removal of CTP) was subjected to three-dimensionalstructural modeling using software phyre2, and then it is found that theGS linker peptide is far from the CDR region (FIG. 2-15) and does notaffect antigen-antibody binding. Even if the GS linker peptide is closeto the CDR region, the GS linker peptide can freely move away from theantigen-antibody binding region due to its own flexibility and thus doesnot affect antigen-antibody binding.

Further, the molecular docking between the anti-CD3 scFv and its antigenCD3 epsilon chain was simulated by software Discovery Studio. Since thestructure of the double-chain anti-CD3 FV is highly similar to thestructure of the anti-CD3 scFv, the structure simulation was performedusing the double-chain anti-CD3 FV instead of the anti-CD3 scFv. Thesimulation results show that the antigen CD3 epsilon chain binds to CDR2and CDR3 of VH of the anti-CD3 Fv while does not bind to the CDR1 region(FIG. 2-16), which indicates that the CTP overlaying the VH CDR1 regionof the anti-CD3 Fv does not interfere with the binding of the anti-CD3scFv to the antigen. However, given that the CD3 molecule is a complexincluding one CD3 gamma chain, one CD3 delta chain, and two CD3 epsilonchains, the CD3 molecule, together with the TCR and Zeta chains,constitutes a T-cell receptor complex. Although the CTP peptide fragmentcovering the VH CDR1 of the anti-CD3 scFv does not directly interferewith the binding of the anti-CD3 scFv to its antigen CD3 epsilon chain,the CTP peptide fragment may indirectly affect the binding of theanti-CD3 scFv to its antigen CD3 epsilon chain by making spatialstructural contact with a certain constituent protein of the T-cellreceptor complex.

Due to the presence of CTP covering the VH CDR1 region of the anti-CD3scFv, the binding affinity of the anti-CD3 scFv for its antigen isgreatly diminished so that there is no substantial release of cytokinescaused by the overactivation of T cells, thereby avoiding someunnecessary T cell-mediated non-specific killing.

Example 3 Pharmacodynamics Study of Anti-Her2×CD3 Bispecific Antibodiesin a Mouse Transplanted Tumor Model 3.1 NCG Mouse Model of TransplantedTumor Constructed by Subcutaneously Co-Inoculating Human CIK Cells andHuman Breast Cancer Cells HCC1954

Her2-positive human breast cancer cells HCC1954 were selected to studythe effect of bispecific antibodies in inhibiting tumor growth in vivoin an NCG mouse model of transplanted tumor constructed bysubcutaneously co-inoculating human CIK cells and HCC1954 cells.

The peripheral blood of a normal human was subjected to density gradientcentrifugation (Lymphoprep™, Lymphocytes Separation Medium, STEMCELL) toseparate human PBMCs. Then the human PBMCs were resuspended in RPMI-1640culture medium added with 10% inactivated FBS, and added with OKT3 at afinal concentration of 1 μg/mL and human IL-2 at 250 IU/mL. After threedays of culture, the human PBMCs were centrifuged at 300 g for 5minutes, and the medium was changed. The cells were cultured inRPMI-1640 added with 10% inactivated FBS and added with human IL-2 at250 IU/mL. After that, a fresh medium was then added every 2 days andCIK cells were collected on the tenth day of culture. Female NCG mice atthe age of seven to eight weeks (purchased from Jiangsu GemPharmatechCo. Ltd Company) were selected and HCC1954 cells in the logarithmicgrowth stage were collected. 5×10⁶ HCC1954 cells and 5×10⁵ CIK cellswere mixed and inoculated subcutaneously on the right back of each NCGmouse. One hour later, the mice were randomly divided into seven groupswith five mice in each group according to their weights andintraperitoneally administered with corresponding drugs. All positivecontrol groups and PBS control group were administered twice a week fora total of 3 doses, wherein the positive control groups wereadministered with Herceptin (from Roche) at doses of 1 mg/kg and 3mg/kg, respectively, and the PBS control group was administered with aPBS solution of the same volume as Herceptin. The treated groups wereadministered with bispecific antibodies AB7K4 and AB7K7 every day atdoses of 0.1 mg/kg and 1 mg/kg, respectively, for a total of 10 doses.The day of administration was recorded as Day 0. The maximum diameter(D) and the minimum diameter (d) of the tumor were measured weekly withan electronic vernier caliper. The volume of the tumor was calculatedusing the following formula: volume (mm³)=[D×d²]/2. The tumor growthinhibition rate (TGI) was calculated for each treated group using thefollowing formula: TGI (%)=(1−volume of the treated group/volume of thecontrol group)×100%.

As shown in FIG. 3-1, on Day 33 of administration, the average tumorvolume of the PBS control group was 1494.61±500.28 mm³; the averagetumor volume of the treated group administrated with Herceptin at a doseof 1 mg/kg was 1327.29±376.65 mm³; the average tumor volume of thetreated group administrated with Herceptin at a dose of 3 mg/kg was510.49±106.07 mm³, and the TGI was 65.84%, which was not significantlydifferent from that of the control group.

The average tumor volumes of treated groups administrated with AB7K4 atdoses of 0.1 mg/kg and 1 mg/kg were 304.10±108.50 mm³ and 79.70±58.14mm³, respectively, and TGIs thereof were 79.65% and 94.67%,respectively, which were significantly different from that of the PBScontrol group (P<0.05). The average tumor volumes of treated groupsadministrated with AB7K7 at doses of 0.1 mg/kg and 1 mg/kg were385.82±95.41 mm³ and 209.98±51.74 mm³, respectively, and TGIs thereofwere 74.19% and 85.95%, respectively, which were significantly differentfrom that of the PBS control group (P<0.05). In summary, the resultsshow that the bispecific antibodies AB7K4 and AB7K7 at different dosescould inhibit the growth of tumor cells by activating human immune cellsin animals and exhibited great anti-tumor effects; and at the same doseof 1 mg/kg, the anti-tumor effect of the bispecific antibody was betterthan that of the monoclonal antibody Herceptin.

3.2 NPG Mouse Model of Transplanted Tumor Constructed by SubcutaneouslyCo-Inoculating Human CIK Cells and Human Breast Cancer Cells HCC1954

Her2-positive human breast cancer cells HCC1954 were selected to studythe inhibiting effect of bispecific antibodies on tumor growth in vivoin an NPG mouse model of transplanted tumor constructed bysubcutaneously co-inoculating human CIK cells and human breast cancercells HCC1954.

CIK cells were prepared in the method as described in Example 3.1.Female NPG mice at the age of seven to eight weeks (purchased fromBeijing Vitalstar Biotechnology Co., Ltd.) were selected and HCC1954cells in the logarithmic growth stage were collected. 5×10⁶ HCC1954cells and 5×10⁵ CIK cells were mixed and inoculated subcutaneously onthe right back of each NPG mouse. After 6 days of tumor growth, the micewere randomly divided into three groups with six mice in each groupaccording to the tumor volumes and weights and intraperitoneallyadministered with corresponding drugs. Specifically, AB7K7 treatedgroups were administered twice a week at doses of 0.1 mg/kg and 1 mg/kg,respectively, and the control group was administered with a PBS solutionof the same volume as AB7K7. The day of administration was recorded asDay 0. The maximum diameter (D) and the minimum diameter (d) of thetumor were measured weekly. The volume (mm³) of the tumor of each groupand the tumor growth inhibition rate (TGI) (%) of each treated groupwere calculated using the formulas as shown in Example 3.1.

As shown in FIG. 3-2, on Day 21 of administration, the average tumorvolume of the PBS control group was 821.73±201.82 mm³; the average tumorvolume of the treated group administrated with AB7K7 at a dose of 0.1mg/kg was 435.60±51.04 mm³, and the TGI was 50.83%, which was notsignificantly different from that of the control group; the averagetumor volume of the treated group administrated with AB7K7 at a dose of1 mg/kg was 40.98±12.64 mm³, and the TGI was 95.37%, which wassignificantly different from that of the control group (P<0.01). Theabove results show that the administration of the bispecific antibodyAB7K7 had a good therapeutic effect even when tumors had grown to acertain volume, wherein 50% tumor inhibition effect was achieved at thelow dose of 0.1 mg/kg, and there was complete tumor regression in 4 of 6mice in the treated group at the dose of 1 mg/kg and the tumor volumesin the other 2 mice were both less than 100 mm³, which was smaller thanthe tumor volume at the time of grouping (the average tumor volume ofthis group at the time of grouping was 161.37±18.98 mm³). Therefore, thebispecific antibody AB7K7 had a great therapeutic effect on tumors.

In addition, the inhibiting effect of bispecific antibodies AB7K7 andAB7K8 on tumor growth in the above-described transplanted tumor model attwo administration frequencies were also studied. CIK cells wereprepared in the method as described above. Female NPG mice at the age ofseven to eight weeks were selected, and 5×10⁶ HCC1954 cells and 5×10⁵CIK cells were mixed and inoculated subcutaneously on the right back ofeach NPG mouse. One hour later, the mice were randomly divided into sixgroups with six mice in each group according to their weights andintraperitoneally administered with corresponding drugs. Specifically,the control group and the Herceptin treated group were administeredtwice a week, wherein Herceptin was administrated at a dose of 3 mg/kgand the control group was administered with a PBS solution of the samevolume as Herceptin. The bispecific antibody AB7K7 was administered at adose of 1 mg/kg and AB7K8 was administered at a dose of 0.7 mg/kg. Twoadministration frequencies were set for each of the two bispecificantibodies, wherein the QD group was administered once a day for 10consecutive days and the BIW group was administered twice a week. Theday of administration was recorded as Day 0. The maximum diameter (D)and the minimum diameter (d) of the tumor were measured weekly. Thetumor volume (mm³) of each group and the tumor growth inhibition rate(TGI) (%) of each treated group were calculated using the formulas asshown above.

As shown in FIG. 3-3, on Day 25 of administration, the average tumorvolume of the PBS control group was 1588.12±120.46 mm³; the averagetumor volume of the treated group administrated with Herceptin at a doseof 3 mg/kg was 361.72±134.70 mm³; the average tumor volumes of the QDgroup and the BIW group administrated with AB7K7 were 260.18±45.96 mm³and 239.39±40.62 mm³, respectively, and TGIs were 83.62% and 84.93%,respectively, which were significantly different from that of the PBScontrol group (P<0.01); the average tumor volumes of the QD group andthe BIW group administrated with AB7K8 were 284.98±26.62 mm³ and647.14±118.49 mm³, respectively, and TGIs were 82.06% and 59.25%,respectively, which were significantly different from that of the PBScontrol group (P<0.01). As can be seen from the above results, theanti-tumor effect of the bispecific antibody AB7K7 was superior to thatof the Herceptin in both the QD group and the BIW group; at equimolardoses, AB7K8 and AB7K7 in the QD group exhibited basically equal tumorinhibiting effects, while the anti-tumor effect of AB7K7 in the BIWgroup was significantly superior than that of AB7K8, presumably due tothe fact that AB7K8 is a bispecific antibody of BiTE configuration withno Fc domain, and therefore AB7K7 has a longer half-life than AB7K8,from which it is anticipated that the clinical administration frequencyof AB7K7 is reduced and AB7K7 has a better therapeutic effect.

3.3 NPG Mouse Model of Transplanted Tumor Constructed by SubcutaneouslyCo-Inoculating Human CIK Cells and Human Ovarian Cancer Cells SK-OV-3

Her2-positive human ovarian cancer cells SK-OV-3 were selected to studythe inhibiting effect of bispecific antibodies on tumor growth in vivoin an NPG mouse model of transplanted tumor constructed bysubcutaneously co-inoculating human CIK cells and SK-OV-3 cells.

The peripheral blood of a normal human was subjected to density gradientcentrifugation to separate human PBMCs. Then the human PBMCs wereresuspended in McCoy's 5A culture medium added with 10% inactivated FBS,and added with OKT3 at a final concentration of 1 μg/mL and human IL-2at 250 IU/mL. After three days of culture, the human PBMCs werecentrifuged at 300 g for 5 minutes, and the supernatant was discarded.The cells were resuspended in RPMI-1640 added with 10% inactivated FBSand added with 250 IU/mL of human IL-2. After that, a fresh medium wasthen added every 2 days and CIK cells were collected on the tenth day ofculture. Female NPG mice at the age of seven to eight weeks wereselected and SK-OV-3 cells (purchased from the cell bank of ChineseAcademy of Sciences, Shanghai) in the logarithmic growth stage werecollected. 3×10⁶ SK-OV-3 cells and 3×10⁵ CIK cells were mixed andinoculated subcutaneously on the right back of each NPG mouse. One hourafter inoculation, the mice were randomly divided into seven groups withsix mice in each group according to their weights and intraperitoneallyadministered with corresponding drugs. Herceptin and AB7K7 treatedgroups were administered twice a week at doses of 1 mg/kg, 0.2 mg/kg,and 0.04 mg/kg, respectively, and the control group was administeredwith a PBS solution of the same volume. The day of administration wasrecorded as Day 0. The maximum diameter (D) and the minimum diameter (d)of the tumor were measured weekly. The volume (mm³) of the tumor of eachgroup and the tumor growth inhibition rate (TGI) (%) of each treatedgroup were calculated using the formulas as shown in Example 3.1.

As shown in FIG. 3-4, on Day 21 of administration, the average tumorvolume of the PBS control group was 834.09±45.64 mm³; the average tumorvolumes of the treated groups administrated with Herceptin at doses of 1mg/kg, 0.2 mg/kg, and 0.04 mg/kg were 644.84±58.22 mm³, 884.95±38.63mm³, and 815.79±78.39 mm³, respectively; and the tumors in all AB7K7treated groups were completely regressed. The above results show that inthe ovarian cancer SK-OV-3 model, AB7K7 enabled the tumor to completelyregress even at a very low dose of 0.04 mg/kg, exhibiting an excellentanti-tumor effect.

3.4 NPG Mouse Model of Transplanted Tumor Constructed by SubcutaneouslyCo-Inoculating Human CIK Cells and Human Colon Cancer Cells HT-29

Her2-positive human colon cancer cells HT-29 were selected to study theinhibiting effect of bispecific antibodies on tumor growth in vivo in anNPG mouse model of transplanted tumor constructed by subcutaneouslyco-inoculating human CIK cells and HT-29 cells.

CIK cells were prepared in the method as described in Example 3.1.Female NPG mice at the age of seven to eight weeks were selected andHT-29 cells (purchased from the cell bank of Chinese Academy ofSciences, Shanghai) in the logarithmic growth stage were collected.3×10⁶ HT-29 cells and 3×10⁶ CIK cells were mixed and inoculatedsubcutaneously on the right back of each NPG mouse. One hour afterinoculation, the mice were randomly divided into five groups with sixmice in each group according to their weights and intraperitoneallyadministered with corresponding drugs. Specifically, Herceptin wasadministered at a dose of 3 mg/kg, and AB7K7 was administered at dosesof 3 mg/kg, 1 mg/kg, and 0.3 mg/kg, respectively. All treated groupswere administered twice a week. The control group was administered witha PBS solution of the same volume. The day of administration wasrecorded as Day 0. The maximum diameter (D) and the minimum diameter (d)of the tumor were measured weekly. The volume (mm³) of the tumor of eachgroup and the tumor growth inhibition rate (TGI) (%) of each treatedgroup were calculated using the formulas as shown in Example 3.1.

As shown in FIG. 3-5, on Day 21 of administration, the average tumorvolume of the PBS control group was 1880.52±338.26 mm³; the averagetumor volume of the treated group administrated with Herceptin at a doseof 3 mg/kg was 1461.36±177.94 mm³; the average tumor volumes of thetreated groups administrated with AB7K7 at doses of 3 mg/kg, 1 mg/kg,and 0.3 mg/kg were 13.94±7.06 mm³, 26.31±10.75 mm³, and 10.47±6.71 mm³,wherein tumors in four mice in the treated group at a dose of 0.3 mg/kgwere completely regressed, tumors in three mice in the treated group ata dose of 1 mg/kg were completely regressed, and tumors in four mice inthe treated group at a dose of 3 mg/kg were completely regressed. Theabove results show that in the colon cancer HT-29 model, Herceptin hadfew pharmacological effect on this tumor model, whereas AB7K7 exhibitedcomplete tumor regression in mice at all three doses and exhibitedexcellent anti-tumor effect even at very low doses.

3.5 CD34 Immune-Reconstituted NPG Mouse Model of Transplanted TumorConstructed by Inoculating Human Breast Cancer Cells HCC1954

Her2-positive human breast cancer cells HCC1954 were selected to studythe inhibiting effect of bispecific antibodies on tumor growth in vivoin a CD34 immune-reconstituted NPG mouse model of transplanted tumorconstructed by subcutaneously inoculating human breast cancer cellsHCC1954.

CD34-positive hematopoietic stem cells were enriched from freshumbilical cord blood using CD34-positive selective magnetic beads(purchased from Miltenyi Biotec, Germany). Female NPG mice at the age ofseven to eight weeks (purchased from Beijing Vitalstar BiotechnologyCo., Ltd.) were selected and injected with CD34-positive hematopoieticstem cells via the tail vein to reconstitute a human immune system ineach mouse. Sixteen weeks later, blood was collected from the orbitalvenous plexus of mice for flow cytometry, and when the proportion ofhuman CD45 in mice was greater than 15%, it was considered that theimmune reconstitution succeeded. HCC1954 cells in the logarithmic growthstage were collected and 5×10⁶ HCC1954 cells were inoculatedsubcutaneously on the right back of the mice with successful immunereconstitution. One hour after inoculation, the mice were randomlydivided into three groups with six mice in each group according to theirweights. The treated groups were intraperitoneally administered withAB7K7 and Herceptin at a dose of 1 mg/kg, and the control group wasadministered with a PBS solution of the same volume, twice a week for atotal of 6 doses. The day of administration was recorded as Day 0. Themaximum diameter (D) and the minimum diameter (d) of the tumor weremeasured weekly. The volume (mm³) of the tumor of each group and thetumor growth inhibition rate (TGI) (%) of each treated group werecalculated using the formulas as shown in Example 3.1.

As shown in FIG. 3-6, on Day 21 of administration, the average tumorvolume of the PBS control group was 475.23±58.82 mm³; the average tumorvolume of the treated group administrated with Herceptin was293.27±66.35 mm³, and the TGI was 38.29%, which was not significantlydifferent from that of the control group; the average tumor volume ofthe treated group administrated with AB7K7 was 0.67±0.67 mm³, and theTGI was 99.86%, meaning that basically all tumors were regressed, whichwas significantly different from that of the control group (P<0.01). Insummary, the above results show that the bispecific antibody AB7K7 hadan excellent anti-tumor effect in the CD34 immune-reconstituted model.

3.6 PBMC Immune-Reconstituted NPG Mouse Model of Transplanted TumorConstructed by Inoculating Human Breast Cancer Cells HCC1954

Her2-positive HCC1954 cells were selected to study the inhibiting effectof bispecific antibodies on tumor growth in vivo in a PBMCimmune-reconstituted NPG mouse model of transplanted tumor constructedby inoculating human breast cancer cells HCC1954.

The peripheral blood of a normal human was subjected to density gradientcentrifugation to separate human PBMCs. Female NPG mice at the age offive to six weeks were selected and intraperitoneally injected withhuman PBMC cells to reconstitute a human immune system in each mouse.After seven days of PBMC injection, HCC1954 cells in the logarithmicgrowth stage were collected and 5×10⁶ HCC1954 cells were inoculatedsubcutaneously on the right back of each mouse. After 13 days of PBMCinjection, blood was collected from the orbital venous plexus for flowcytometry, and when the proportion of human CD45 in mice was greaterthan 15%, it was considered that the immune reconstitution succeeded.After 14 days of PBMC injection, the mice with successful immunereconstitution were randomly divided into two groups with six mice ineach group according to the tumor volumes and weights. The treated groupwas intraperitoneally administered with AB7K7 at a dose of 1 mg/kg, andthe control group was administered with PBS, three times a week. The dayof administration was recorded as Day 0. The maximum diameter (D) andthe minimum diameter (d) of the tumor were measured weekly. The volume(mm³) of the tumor of each group and the tumor growth inhibition rate(TGI) (%) of each treated group were calculated using the formulas asshown in Example 3.1.

As shown in FIG. 3-7, on Day 23 of administration, the average tumorvolume of the PBS control group was 1224.05±224.39 mm³; the averagetumor volume of the treated group administrated with AB7K7 was32.00±0.00 mm³, and the TGI was 97.41%, meaning that basically alltumors were regressed, which was significantly different from that ofthe control group (P<0.001). In summary, the above results show that thebispecific antibody AB7K7 had an excellent anti-tumor effect in the PBMCimmune-reconstituted model.

Example 4 Evaluation of the Safety of Anti-Her2×CD3 BispecificAntibodies

4.1 Bispecific Antibodies being Incapable of Mediating Non-SpecificKilling on Her2-Negative Tumor Cells

Her2-negative human Burkkit's lymphoma Raji cells were selected to studywhether bispecific antibodies can inhibit tumor growth in an NCG mousemodel of transplanted tumor constructed by subcutaneously co-inoculatinghuman CIK cells and human Burkkit's lymphoma Raji cells.

CIK cells were prepared in the method as described in Example 3.1.Female NCG mice at the age of seven to eight weeks were selected andRaji cells (purchased from the cell bank of Chinese Academy of Sciences,Shanghai) in the logarithmic growth stage were collected. 5×10⁶ Rajicells and 2×10⁶ CIK cells were mixed and inoculated subcutaneously onthe right back of each NCG mouse. One hour after inoculation, the micewere randomly divided into three groups with five mice in each groupaccording to their weights. The treated groups were intraperitoneallyadministered with AB7K4 and AB7K7 at a dose of 1 mg/kg, and the controlgroup was administered with a PBS solution of the same volume, once aday continuously for 10 days. The day of administration was recorded asDay 0. The maximum diameter (D) and the minimum diameter (d) of thetumor were measured weekly. The volume (mm³) of the tumor of each groupand the tumor growth inhibition rate (TGI) (%) of each treated groupwere calculated using the formulas as shown in Example 3.1.

As shown in FIG. 4-1, on Day 25 of administration, the average tumorvolume of the PBS control group was 2439.88±193.66 mm³; the averagetumor volume of the treated group administrated with AB7K4 was2408.81±212.44 mm³; the average tumor volume of the treated groupadministrated with AB7K7 was 2598.11±289.35 mm³; and there was nodifference between the average tumor volume of each of the two treatedgroups and the average tumor volume of the control group. In summary,the results show that bispecific antibodies AB7K4 and AB7K7 exhibited nonon-specific killing on Her2-negative cell strains, which indicates thatAB7K4 and AB7K7 do not mediate T cells to kill non-target tissues invivo (i.e., specifically dependent on binding of bispecific antibodiesto corresponding target antigens), there is no off-target toxicity, andthe safety is high.

4.2 Bispecific Antibodies Killing Tumor Cells Depending on theActivation of T Cells

Her2-positive human breast cancer cells HCC1954 were selected to studywhether bispecific antibodies inhibit tumor growth in an NPG mouse modelof transplanted tumor constructed by subcutaneously inoculating humanbreast cancer cells HCC1954.

Female NPG mice at the age of seven to eight weeks were selected andHCC1954 cells in the logarithmic growth stage were collected. 5×10⁶HCC1954 cells and Matrigel (Corning, Cat. No. 354234) were mixed in avolume ratio of 1:1 and then inoculated subcutaneously on the right backof each NPG mouse. After 6 days of tumor growth, the mice were randomlydivided into three groups with six mice in each group according to thetumor volumes and weights. The treated groups were intraperitoneallyadministered with Herceptin at a dose of 3 mg/kg and AB7K7 at a dose of1 mg/kg, respectively, and the control group was administered with a PBSsolution of the same volume, twice a week. The day of administration wasrecorded as Day 0. The maximum diameter (D) and the minimum diameter (d)of the tumor were measured weekly. The volume (mm³) of the tumor of eachgroup and the tumor growth inhibition rate (TGI) (%) of each treatedgroup were calculated using the formulas as shown in Example 3.1.

As shown in FIG. 4-2, on Day 21 of administration, the average tumorvolume of the PBS control group was 1311.35±215.70 mm³; the averagetumor volume of the treated group administrated with Herceptin was273.98±60.10 mm³; the average tumor volume of the treated groupadministrated with AB7K7 was 1243.20±340.31 mm³, which was not differentfrom the average tumor volume of the control group. In summary, theresults show that AB7K7 did not inhibit the growth of HCC1954subcutaneous tumors in the absence of human immune cells, indicatingthat bispecific antibody AB7K7 needs to be mediated by immune effectorcells so as to kill tumor cells, unlike Herceptin, which primarilydepends on FcγR-mediated ADCC or CDC effects to kill tumor cells. Thus,it is proved that Fc variants contained in AB7K7 cannot bind to FcγR,which avoids mediating systemic activation of T cells caused byextensive expression of its receptor FcγR, resulting in higher drugsafety.

4.3 Evaluation of Toxicity of Bispecific Antibodies to Normal CynomolgusMonkeys

Adult cynomolgus monkeys (purchased from Guangzhou XiangguanBiotechnology Co., Ltd.) at the age of 3-4 years and with the weight of3-4 kg were divided into three groups with one monkey in each group,wherein the three groups were a vehicle control group, an AB7K7 treatedgroup and an AB7K8 treated group. The groups were administrated viaintravenous drip by a peristaltic pump for 1 hour on Day 0 (D0), Day 7(D7), Day 21 (D21), and Day 28 (D28), respectively, for a total of fourdoses, and the drug dose was gradually increased each time. The monkeyswere weighed weekly. The dose amount and volume administered are shownin Table 4-1.

On D0, after administration, cynomolgus monkeys in the AB7K8 treatedgroup exhibited somnolence and pupil contraction and recovered to normalthe next day while there was no abnormality in the other groups. On D7,after administration, cynomolgus monkeys in the AB7K7 treated groupexhibited vomiting symptoms 2-3 hours after administration and recoveredto normal the next day of administration while there was no abnormalityin the other groups. On D21, after administration, cynomolgus monkeys inthe AB7K7 treated group exhibited symptoms of vomiting food 3 hoursafter administration and excreted jelly-like feces, cynomolgus monkeysin the AB7K8 treated group exhibited symptoms of vomiting food 1 hourafter administration, and cynomolgus monkeys in both groups recovered tonormal on the second day after administration; On D28, afteradministration, cynomolgus monkeys in both AB7K7 treated group and AB7K8treated group exhibited vomiting symptoms 40 to 50 minutes later andexcreted feces 3 hours later, in which jelly-like mucus was found;cynomolgus monkeys in the AB7K7 treated group excreted watery feces withfishy smelling; and 24 hours later, all the animals recovered to normaland ingested normally. The body weight change of cynomolgus monkeys isshown in FIG. 4-3, wherein the arrow represents the administration time.It can be seen that the body weight of each group does not change toomuch and fluctuates within the normal physiological range.

TABLE 4-1 Dosing schedule for cynomolgus monkey acute toxicityevaluation To-be-tested Group drugs name Dose volume Dose amount G1Vehicle D0: 5 mL/kg N/A control D7: 5 mL/kg group D21: 10 mL/kg D28: 10mL/kg G2 AB7K7 D0: 5 mL/kg D0: 0.06 mg/kg D7: 5 mL/kg D7: 0.3 mg/kg D21:10 mL/kg D21: 1.5 mg/kg D28: 10 mL/kg D28: 3 mg/kg G3 AB7K8 D0: 5 mL/kgD0: 0.04 mg/kg D7: 5 mL/kg D7: 0.2 mg/kg D21: 10 mL/kg D21: 1 mg/kg D28:10 mL/kg D28: 2 mg/kg

The different degree of diarrhea observed in this example may be relatedto the expression of related receptors in the gut, which is supposed tobe caused by the imbalance of chloride ion in the gut caused by theinhibition of heterodimer of Her1/Her2 or Her2/Her3 by bispecificantibodies, which belongs to the extension of pharmacological action andcan recover to normal after 24 hours of administration. Cynomolgusmonkeys were still well tolerated when administrated with AB7K7 at ahigh dose of 3 mg/kg. The results of pharmacodynamics test in mice showthat AB7K7 at a low dose shows a good anti-tumor effect, which indicatesthat AB7K7 has a wide treatment window and high safety.

Example 5 Pharmacokinetics Study of Anti-Her2×CD3 Antibodies 5.1 In VivoPharmacokinetics Test of Bispecific Antibody AB7K7 in SD Mice

AB7K7 was administered to four healthy Sprague-Dawley (SD) rats(purchased from Shanghai Salccas Laboratory Animals Co., Ltd.,) via thetail vein at a dose of 1 mg/kg. The blood sampling time points were Hour1, Hour 3, Hour 6, Hour 24, Hour 72, Hour 96, Hour 120, Hour 168, Hour216 and Hour 264, respectively. A certain amount of whole blood wastaken at each time point, the serum was separated, and then the drugconcentration in the serum was detected by two ELISA methods.

Method I. Plates were coated with the anti-AB7K7 antibody A (AmpsourceBiopharma Shanghai Inc., mouse-anti-herceptin) at a concentration of 0.5μg/mL. AB7K7 was formulated at concentrations of 100 ng/mL, 50 ng/mL, 25ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.125 ng/mL and 1.56 ng/mL, separately.Standard curves were established. HRP-labeled anti-AB7K7 antibody B(Ampsource Biopharma Shanghai Inc., anti-herceptin-HRP) was used at aconcentration of 1:5000, and developed with TMB. The pharmacokineticsparameters were calculated using software PKSolver. Specific parametersare shown in Table 5-1.

Method II. The drug concentration in the serum of the SD rats wasdetected. Plates were coated with the anti-AB7K7 antibody A (AmpsourceBiopharma Shanghai Inc., mouse-anti-herceptin) at a concentration of 0.5μg/mL. AB7K7 was formulated at concentrations of 5 ng/mL, 2.5 ng/mL,1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, 0.156 ng/mL and 0.078 ng/mL,separately. Standard curves were established. Mouse anti-human IgGFc-HRP (Ampsource Biopharma Shanghai Inc.) was added at a concentrationof 1:5000, and developed with TMB. The pharmacokinetics parameters werecalculated using software PKSolver. Specific parameters are shown inTable 5-2.

FIG. 5-1 shows the blood drug concentration of AB7K7 in the body of ratsdetected using two different detection methods. It can be seen that theblood drug concentrations obtained by detecting the concentration ofAB7K7 in blood using two different detection methods were basically thesame, and the calculated pharmacokinetics parameters were roughlyequivalent, which indicates that AB7K7 can be metabolized in the form ofintact molecules in vivo, thereby ensuring the biological function ofAB7K7.

TABLE 5-1 Pharmacokinetics parameters of bispecific antibody AB7K7 in SDrats (Method 1) AUC 0-inf_ob Vz_obs Cl_obs AB7K7 t_(1/2) (h) (ng/mL*h)(μg)/(ng/mL) (μg)/(ng/mL)/h Pharmacokinetics 42.10 550236.77 0.023513.811E−4 parameter

TABLE 5-2 Pharmacokinetics parameters of bispecific antibody AB7K7 in SDrats (Method 2) AUC 0-inf_ob Vz_obs Cl_obs AB7K7 t_(1/2) (h) (ng/mL*h)(μg)/(ng/mL) (μg)/(ng/mL)/h Pharmacokinetics 41.02 706126.89 0.017202.899E−4 parameter

5.2 In Vivo Pharmacokinetics Test on Bispecific Antibody AB7K7 in NPGModel Mice

NPG mice (purchased from Beijing Vitalstar Biotechnology Co., Ltd.) wereinoculated with HCC1954 cells (purchased from the Institute of Cells,Chinese Academy of Sciences) one week before administration, with aninoculum density of 3.5×10⁶/mouse. CIK cells were resuscitated two daysbefore administration, cultured for 24 hours and then collected andinjected intravenously into mice. The mice were randomly divided intothree groups with four mice in each group. The three treated groups wereadministrated at doses of 0.3 mg/kg, 1 mg/kg and 3 mg/kg, respectively.The blood sampling time points were Hour 1, Hour 3, Hour 6, Hour 24,Hour 48, Hour 72, Hour 96, Hour 120, Hour 168, Hour 216 and Hour 264,respectively. A certain amount of whole blood was taken at each timepoint, the serum was separated, and then the drug concentration in theserum was detected by ELISA.

Plates were coated with the anti-AB7K7 antibody A (Ampsource BiopharmaShanghai Inc., mouse-anti-herceptin) at a concentration of 0.5 μg/mL.AB7K7 was formulated at concentrations of 100 ng/mL, 50 ng/mL, 25 ng/mL,12.5 ng/mL, 6.25 ng/mL, 3.125 ng/mL and 1.56 ng/mL, separately. Standardcurves were established. HRP-labeled anti-AB7K7 antibody B (AmpsourceBiopharma Shanghai Inc., mouse-anti-herceptin) was used at aconcentration of 1:5000, and developed with TMB. The pharmacokineticsparameters were calculated using software PKSolver. Specific parametersare shown in Table 5-3. It can be seen from Table 5-3 that thepharmacokinetics parameters of AB7K7 in NPG model mice were notsignificantly different from those in SD rats.

TABLE 5-3 Pharmacokinetics parameters of bispecific antibody AB7K7 inNPG model mice Parameter AUC0-inf_obs Vz_ob Cl_obs Group t_(1/2) (h)(ng/mL*h) (μg)/(ng/mL) (μg)/(ng/mL)/h 0.3 mg/kg IV 39.54 79932.390.004872 8.968E−05 1 mg/kg IV 42.70 597036.63 0.002461 3.996E−05 3 mg/kgIV 46.03 2171649.41 0.002292 3.469E−05

5.3 In Vivo Pharmacokinetics Test on Bispecific Antibody AB7K8 in SDRats

AB7K8 was administered to three healthy SD rats via the tail vein atdoses of 1 mg/kg and 3 mg/kg, respectively. The blood sampling timepoints were Hour 0.25, Hour 0.5, Hour 1, Hour 2, Hour 3, Hour 4, Hour 5,and Hour 7, respectively. A certain amount of whole blood was taken ateach time point, the serum was separated, and then the drugconcentration in the serum was detected by ELISA.

Plates were coated with the anti-AB7K8 antibody C (Ampsource BiopharmaShanghai Inc., mouse-anti-herceptin) at a concentration of 2.5 μg/mL.AB7K8 was formulated at concentrations of 25 ng/mL, 12.5 ng/mL, 6.25ng/mL, 3.125 ng/mL, 1.56 ng/mL, and 0.78 ng/mL, separately. Standardcurves were established. HRP-labeled anti-his antibody (AmpsourceBiopharma Shanghai Inc.) was used at a concentration of 1:5000, anddeveloped with TMB. The pharmacokinetics parameters were calculatedusing software PKSolver. Specific parameters are shown in Table 5-4.

The pharmacokinetics parameter T_(1/2) of AB7K8 were almost the same attwo doses, which indicates that AB7K8 showed linear metabolic kineticsin SD rats. Since AB7K8 does not contain Fc, T_(1/2) of AB7K8 is veryshort, about twenty times shorter than T_(1/2) of AB7K7.

TABLE 5-4 Pharmacokinetics parameters of bispecific antibody AB7K8 in SDrats AUC 0-inf_ob Vz_obs Cl_obs AB7K8 t_(1/2) (h) (ng/mL*h) (μg)/(ng/mL)(μg)/(ng/mL)/h 1 mg/kg IV 2.27 4623.14 0.17082 0.05191 3 mg/kg IV 1.9820608.77 0.10220 0.03579

5.4 In Vivo Pharmacokinetics Test on Bispecific Antibody AB7K in SD Rats

AB7K was administered to four healthy SD rats via the tail vein at adose of 0.8 mg/kg. The blood sampling time points were Hour 2, Hour 24,Hour 48, Hour 72, Hour 96, Hour 120, Hour 144, Hour 168, Hour 216 andHour 264, respectively. A certain amount of whole blood was taken ateach time point, the serum was separated, and then the drugconcentration in the serum was detected by two ELISA methods.

Method I. Plates were coated with the anti-AB7K antibody A (AmpsourceBiopharma Shanghai Inc., mouse-anti-herceptin) at a concentration of 1μg/mL. AB7K was formulated at concentrations of 20 ng/mL, 10 ng/mL, 5ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL and 0.3125 ng/mL, separately.Standard curves were established. 25 ng/mL of biotin-labeled humanCD3E&CD3D (Acro, Cat. No. CDD-H82W0) was added, incubated for 1 hour,and after that, HRP-labeled streptavidin (BD Pharmingen, Cat. No.554066) diluted at a factor of 1:500 was added, and developed with TMB.The pharmacokinetics parameters were calculated using software PKSolver.Specific parameters are shown in Table 5-5.

TABLE 5-5 Pharmacokinetics parameters of bispecific antibody AB7K AUC0-inf_ob Vz_obs Cl_obs AB7K t_(1/2) (h) (ng/mL*h) (μg)/(ng/mL)(μg)/(ng/mL)/h Pharmacokinetics 60.47 1022788.69 0.01726 1.985E−4parameter

Method II. Plates were coated with the anti-AB7K antibody A (AmpsourceBiopharma Shanghai Inc., mouse-anti-herceptin) at a concentration of 1μg/mL. AB7K was formulated at concentrations of 20 ng/mL, 10 ng/mL, 5ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL and 0.3125 ng/mL, separately.Standard curves were established. Mouse anti-human IgG Fc-HRP (dilutedat 1:10000) (Ampsource Biopharma Shanghai Inc.) was added, incubated inan incubator for 1 hour, and developed with TMB.

FIG. 5-2 shows the blood drug concentration of AB7K in rats by using twodifferent detection methods. From the results, it is showed that thedifference between the two detection methods is large. Theconcentrations of the first two points (2h, 1D) on the curve were close,but after the next day, the concentrations detected by the two methodsdiffered greatly, which is supposed to be caused by the fact that thelinkage peptide between the heavy chains of anti-CD3 scFv and anti-Her2antibodies was broken. AB7K is structurally unstable in vivo and thuscan't play its biological function, while the improved AB7K7 canmetabolize in complete form in vivo and thus can play its biologicalfunction normally.

5.5 In Vivo Pharmacokinetics Test on Bispecific Antibodies AB7K7 andAB7K8 in Cynomolgus Monkeys

Female cynomolgus monkeys (purchased from Guangzhou XiangguanBiotechnology Co., Ltd.) with the weight of 3-4 kg were divided intothree groups with one monkey in each group. The first group (G1-1) was ablank control group; the second group (G2-1) was an AB7K7 treated groupadministrated at a dose of 0.3 mg/kg; and the third group (G3-1) was anAB7K8 treated group administrated at a dose of 0.2 mg/kg. The bloodsampling time points were Minute 15, Hour 1, Hour 3, Hour 6, Hour 24,Hour 48, Hour 72, Hour 96, Hour 144, Hour 192, Hour 240 and Hour 288,respectively, a total of 13 time points. Serum was collected from bloodand frozen at −80° C. The concentration of the drug in serum wasdetermined by ELISA.

Plates were coated with the anti-AB7K7 antibody A (Ampsource BiopharmaShanghai Inc., mouse-anti-herceptin) at a concentration of 0.5 μg/mL.AB7K7 was formulated at concentrations of 100 ng/mL, 50 ng/mL, 25 ng/mL,12.5 ng/mL, 6.25 ng/mL, 3.125 ng/mL and 1.56 ng/mL, separately. Standardcurves were established. HRP-labeled anti-AB7K7 antibody B (AmpsourceBiopharma Shanghai Inc., mouse-anti-herceptin) was used at aconcentration of 1:5000, and developed with TMB. The pharmacokineticsparameters were calculated using software PKSolver. Specific parametersare shown in Table 5-6.

FIG. 5-3 shows the blood drug concentration of AB7K7 in rats. T_(1/2) ofAB7K7 in the normal cynomolgus monkey was only about eight hours.Pharmacokinetics parameters of AB7K8 could not be calculated due to toofew points on the concentration-time curve of AB7K8. However, it can beseen from the concentration-time curve that the half-life of AB7K7 inthe normal cynomolgus monkey was much longer than the half-life ofAB7K8.

TABLE 5-6 Pharmacokinetics parameters of bispecific antibody AB7K7 incynomolgus monkeys AUC 0-inf_obs Vz_obs Cl_obs AB7K7 t_(1/2) (h)(ng/mL*h) (μg)/(ng/mL) (μg)/(ng/mL)/h Pharmacokinetics 7.95 87995.480.1563 0.01364 parameter

5.6 Evaluation of Abilities of Bispecific Antibodies to Bind to FcRn byELISA

Each bispecific antibody was diluted with the PBS solution to aconcentration of 10 μg/ml and added to 96-well plates, 100 μl per well.The plates were coated at 4° C. overnight. The plates were then blockedwith 1% skimmed milk powder for 1 hour at room temperature.Biotin-labeled FcRn proteins (ACRO Biosystem, Cat. No. FCM-H8286) werediluted using diluents at pH of 6.0 and 7.0, respectively, with a 4-foldgradient for a total of 11 concentration gradients. The 96-well plateswere then washed with PBST of the same pH, and then each of thebispecific antibodies diluted with the diluent at the same pH was added.Control wells without antibodies were set. Incubated for 1 hour at roomtemperature. Plates were washed with the PBST solution of the same pH,streptavidin-HRP (BD, Cat. No. 554066) was added to 96-well plates, 100μl per well, and the plates were incubated for 0.5 hours at roomtemperature. 96-well plates were washed with PBST, and TMB was added tothe plates, 100 μl per wells. Color development was performed at roomtemperature for 15 minutes, and then 0.2 M H₂SO₄ was added to stop thecolor development reaction. The light absorbance values at A450-620 nmwere measured by a microplate reader. Analysis was performed by softwareOriginPro 8, and the EC₅₀ values for the binding of bispecificantibodies to FcRn were calculated.

The results show that the ability of each antibody to bind to FcRn wasdifferent under different pH conditions, and it is analyzed inconjunction with data of in vivo PK that the half-life of bispecificantibody AB7K7 was longer than the half-life of AB7K but shorter thanthe half-life of Herceptin, which may be more favorable for clinicalapplication (FIGS. 5-4 and 5-5). Table 5-7 and Table 5-8 show thedetection results of the ability of each antibody to bind to FcRn at pHs6.0 and 7.0, respectively.

TABLE 5-7 Detection of abilities of bispecific antibodies AB7K, AB7K5and AB7K7 to bind to FcRn at pH 6.0 Herceptin AB7K AB7K5 AB7K7 EC₅₀(μg/ml) 2.591 0.8027 1.706 0.4630

TABLE 5-8 Detection of abilities of bispecific antibodies AB7K, AB7K5and AB7K7 to bind to FcRn at pH 7.0 Herceptin AB7K AB7K5 AB7K7 EC₅₀(μg/ml) −287.1 1.651 13.43 4.838

Example 6 Preparation of Bispecific Antibody with scFv1-scFv2-FcConfiguration

According to the above research results of six kinds of anti-Her2×CD3bispecific antibodies, it can be determined that the bispecific antibodywith scFv1-scFv2-Fc configuration such as AB7K7 is easy to be prepared,can be purified in a simple and efficient method, and has greatstability in preparation and storage process. More advantageously, sucha bispecific antibody has a weak non-specific killing effect on normalcells, has significant advantages of controlled toxic and side effectspossibly caused by overactivation of effector cells, and has gooddruggability.

With reference to the design and preparation method of the bispecificantibody AB7K7 in Example 1, a series of bispecific antibody moleculesthat target immune effector cell antigen CD3 and a tumor-associatedantigen were constructed. Such bispecific antibody molecules aretetravalent homodimers formed by two identical polypeptide chains thatbind to each other by an interchain disulfide bond in the hing region ofthe Fc fragment, wherein each polypeptide chain consists of, in sequencefrom N-terminus to C-terminus, an anti-TAA scFv, a linker peptide, ananti-CD3 scFv, and an Fc fragment. The molecular composition of eachstructural unit of each bispecific antibody is described below indetail.

The tumor-associated antigen includes, but is not limited to, CD19,CD20, CD22, CD25, CD30, CD33, CD38, CD39, CD40, CD47, CD52, CD73, CD74,CD123, CD133, CD138, BCMA, CA125, CEA, CS1, DLL3, DLL4, EGFR, EpCAM,FLT3, gpA33, GPC-3, Her2, MEGE-A3, NYESO1, PSMA, TAG-72, CIX,folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR2, VEGFR3, Cadherin,Integrin, Mesothelin, Claudin18, αVβ3, α5β1, ERBB3, c-MET, IGF1R, EPHA3,TRAILR1, TRAILR2, RANKL, B7 protein family, Mucin family, FAP, andTenascin; preferably, the tumor-associated antigen is CD19, CD20, CD22,CD30, CD38, BCMA, CS1, EpCAM, CEA, Her2, EGFR, CA125, Mucin1, GPC-3, andMesothelin.

Some preferred amino acid sequences of the VH domain and itscomplementary determining regions (HCDR1, HCDR2, and HCDR3) and aminoacid sequences of the VL domain and its complementary determiningregions (LCDR1, LCDR2 and LCDR3) of a first single-chain Fv targetingthe tumor-associated antigen are exemplified in Table 6-1, wherein theamino acid composition of the linker peptide between VH and VL of theanti-TAA scFv is (GGGGS)_(n), wherein n=1, 2, 3, 4 or 5.

TABLE 6-1 Amino acid sequences of the anti-TAA scFv includedin the bispecific antibody and amino acid sequences of its CDR regionsCD19 SEQ ID NO: 9 HCDR1 SYWMN SEQ ID NO: 10 HCDR2 QIWPGDGDTNYNGKFKGSEQ ID NO: 11 HCDR3 RETTTVGRYYYAMDY SEQ ID NO: 12 LCDR1 KASQSVDYDGDSYLNSEQ ID NO: 13 LCDR2 DASNLVS SEQ ID NO: 14 LCDR3 QQSTEDPWTQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMN SEQ ID NO: 15 VHWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATL TADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSS DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDS SEQ ID NO: 16 VLYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSG SGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIK CD19 SEQ ID NO: 17 HCDR1 SNWMH SEQ ID NO: 18 HCDR2EIDPSDSYTNYNQNFQG SEQ ID NO: 19 HCDR3 GSNPYYYAMDY SEQ ID NO: 20 LCDR1SASSGVNYMH SEQ ID NO: 21 LCDR2 DTSKLAS SEQ ID NO: 22 LCDR3 HQRGSYTQVQLVQPGAEVVKPGASVKLSCKTSGYTFTSNWMH SEQ ID NO: 23 VHWVKQAPGQGLEWIGEIDPSDSYTNYNQNFQGKAKL TVDKSTSTAYMEVSSLRSDDTAVYYCARGSNPYYYAMDYWGQGTSVTVSS SEQ ID NO: 24 VL EIVLTQSPAIMSASPGERVTMTCSASSGVNYMHWYQQKPGTSPRRWIYDTSKLASGVPARFSGSGSGTDY SLTISSMEPEDAATYYCHQRGSYTFGGGTKLEIKCD19 SEQ ID NO: 25 HCDR1 TSGMGVG SEQ ID NO: 26 HCDR2 HIWWDDDKRYNPALKSSEQ ID NO: 27 HCDR3 MELWSYYFDY SEQ ID NO: 28 LCDR1 SASSSVSYMHSEQ ID NO: 29 LCDR2 DTSKLAS SEQ ID NO: 30 LCDR3 FQGSVYPFT SEQ ID NO: 31VH QVQLQESGPGLVKPSQTLSLTCTVSGGSISTSGMGVGWIRQHPGKGLEWIGHIWWDDDKRYNPALKSRVT ISVDTSKNQFSLKLSSVTAADTAVYYCARMELWSYYFDYWGQGTLVTVSS SEQ ID NO: 32 VL EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQAPRLLIYDTSKLASGIPARFSGSGSGTDF TLTISSLEPEDVAVYYCFQGSVYPFTFGQGTKLEIK CD19 SEQ ID NO: 33 HCDR1 SSWMN SEQ ID NO: 34 HCDR2 RIYPGDGDTNYNVKFKGSEQ ID NO: 35 HCDR3 SGFITTVRDFDY SEQ ID NO: 36 LCDR1 RASESVDTFGISFMNSEQ ID NO: 37 LCDR2 EASNQGS SEQ ID NO: 38 LCDR3 QQSKEVPFT SEQ ID NO: 39VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSSWMNWVRQAPGKGLEWVGRIYPGDGDTNYNVKFKGRFTI SRDDSKNSLYLQMNSLKTEDTAVYYCARSGFITTVRDFDYWGQGTLVTVSS SEQ ID NO: 40 VL EIVLTQSPDFQSVTPKEKVTITCRASESVDTFGISFMNWFQQKPDQSPKLLIHEASNQGSGVPSRFSGSG SGTDFTLTINSLEAEDAATYYCQQSKEVPFTFGGGTKVEIK CD20 SEQ ID NO: 41 HCDR1 SYNMH SEQ ID NO: 42 HCDR2AIYPGNGDTSYNQKFKG SEQ ID NO: 43 HCDR3 STYYGGDWYFNV SEQ ID NO: 44 LCDR1RASSSVSYIH SEQ ID NO: 45 LCDR2 ATSNLAS SEQ ID NO: 46 LCDR3 QQWTSNPPTSEQ ID NO: 47 VH QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATL TADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA SEQ ID NO: 48 VL QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSY SLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK CD20 SEQ ID NO: 49 HCDR1 NYYIH SEQ ID NO: 50 HCDR2 WIYPGDGNTKYNEKFKGSEQ ID NO: 51 HCDR3 DSYSNYYFDY SEQ ID NO: 52 LCDR1 RASSSVSYMHSEQ ID NO: 53 LCDR2 APSNLAS SEQ ID NO: 54 LCDR3 QQWSFNPPT SEQ ID NO: 55VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYIHWVRQAPGQGLEWIGWIYPGDGNTKYNEKFKGRATL TADTSTSTAYLELSSLRSEDTAVYYCARDSYSNYYFDYWGQGTLVTVSS SEQ ID NO: 56 VL DIQMTQSPSSLSASVGDRVTITCRASSSVSYMHWYQQKPGKAPKPLIYAPSNLASGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQQWSFNPPTFGQGTKVEIK CD20 SEQ ID NO: 57 HCDR1 YSWIN SEQ ID NO: 58 HCDR2 RIFPGDGDTDYNGKFKGSEQ ID NO: 59 HCDR3 NVFDGYWLVY SEQ ID NO: 60 LCDR1 RSSKSLLHSNGITYLYSEQ ID NO: 61 LCDR2 QMSNLVS SEQ ID NO: 62 LCDR3 AQNLELPYT SEQ ID NO: 63VH QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTI TADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS SEQ ID NO: 64 VL DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGS GSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIK CD20 SEQ ID NO: 65 HCDR1 DYAMH SEQ ID NO: 66 HCDR2TISWNSGSIGYADSVKG SEQ ID NO: 67 HCDR3 DIQYGNYYYGMDV SEQ ID NO: 68 LCDR1RASQSVSSYLA SEQ ID NO: 69 LCDR2 DASNRAT SEQ ID NO: 70 LCDR3 QQRSNWPITSEQ ID NO: 71 VH EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTI SRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSS SEQ ID NO: 72 VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTD FTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK CD22 SEQ ID NO: 73 HCDR1 RSWMN SEQ ID NO: 74 HCDR2 RIYPGDGDTNYSGKFKGSEQ ID NO: 75 HCDR3 DGSSWDWYFDV SEQ ID NO: 76 LCDR1 RSSQSIVHSVGNTFLESEQ ID NO: 77 LCDR2 KVSNRFS SEQ ID NO: 78 LCDR3 FQGSQFPYT SEQ ID NO: 79VH EVQLVESGGGLVQPGGSLRLSCAASGYEFSRSWMNWVRQAPGKGLEWVGRIYPGDGDTNYSGKFKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCARDGSSWDWYFDVWGQGTLVTVSS SEQ ID NO: 80 VL DIQMTQSPSSLSASVGDRVTITCRSSQSIVHSVGNTFLEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGS GSGTDFTLTISSLQPEDFATYYCFQGSQFPYTFGQGTKVEIK CD22 SEQ ID NO: 81 HCDR1 IYDMS SEQ ID NO: 82 HCDR2YISSGGGTTYYPDTVKG SEQ ID NO: 83 HCDR3 HSGYGTHWGVLFAY SEQ ID NO: 84 LCDR1RASQDISNYLN SEQ ID NO: 85 LCDR2 YTSILHS SEQ ID NO: 86 LCDR3 QQGNTLPWTSEQ ID NO: 87 VH EVQLVESASTGGGLVKPGGSLKLSCAASGFAFSIYDMSWVRQTPEKCLEWVAYISSGGGTTYYPDTVKGR FTISRDNAKNTLYLQMSSLKSEDTAMYYCARHSGYGTHWGVLFAYWGQGTLVT SEQ ID NO: 88 VL DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTD YSLTISNLEQEDFATYFCQQGNTLPWTFGCGTKLEIK CD30 SEQ ID NO: 89 HCDR1 DYYIT SEQ ID NO: 90 HCDR2 WIYPGSGNTKYNEKFKGSEQ ID NO: 91 HCDR3 YGNYWFAY SEQ ID NO: 92 LCDR1 KASQSVDFDGDSYMNSEQ ID NO: 93 LCDR2 AASNLES SEQ ID NO: 94 LCDR3 QQSNEDPWT SEQ ID NO: 95VH QIQLQQSGPEVVKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWIYPGSGNTKYNEKFKGKATL TVDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWFAYWGQGTQVTVSA SEQ ID NO: 96 VL DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPPKVLIYAASNLESGIPARFSGSG SGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIK CD30 SEQ ID NO: 97 HCDR1 AYYWS SEQ ID NO: 98 HCDR2DINHGGGTNYNPSLKS SEQ ID NO: 99 HCDR3 LTAY SEQ ID NO: 100 LCDR1RASQGISSWLT SEQ ID NO: 101 LCDR2 AASSLQS SEQ ID NO: 102 LCDR3 QQYDSYPITSEQ ID NO: 103 VH QVQLQQWGAGLLKPSETLSLTCAVYGGSFSAYYWSWIRQPPGKGLEWIGDINHGGGTNYNPSLKSRVTIS VDTSKNQFSLKLNSVTAADTAVYYCASLTAYWGQGSLVTVSS SEQ ID NO: 104 VL DIQMTQSPTSLSASVGDRVTITCRASQGISSWLTWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYDSYPITFGQGTRLEIK EpCAM SEQ ID NO: 105 HCDR1 SYGMH SEQ ID NO: 106 HCDR2VISYDGSNKYYADSVKG SEQ ID NO: 107 HCDR3 DMGWGSGWRPYYYYGMDV SEQ ID NO: 108LCDR1 RTSQSISSYLN SEQ ID NO: 109 LCDR2 WASTRES SEQ ID NO: 110 LCDR3QQSYDIPYT SEQ ID NO: 111 VH EVQLLESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTI SRDNSKNTLYLQMNSLRAEDTAVYYCAKDMGWGSGWRPYYYYGMDVWGQGTTVTVSS SEQ ID NO: 112 VLELQMTQSPSSLSASVGDRVTITCRTSQSISSYLNW YQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDSATYYCQQSYDIPYTFGQGTKLE IK EpCAM SEQ ID NO: 113 HCDR1 NYGMNSEQ ID NO: 114 HCDR2 WINTYTGESTYADSFKG SEQ ID NO: 115 HCDR3 FAIKGDYSEQ ID NO: 116 LCDR1 RSTKSLLHSNGITYLY SEQ ID NO: 117 LCDR2 QMSNLASSEQ ID NO: 118 LCDR3 AQNLEIPRT SEQ ID NO: 119 VHEVQLVQSGPGLVQPGGSVRISCAASGYTFTNYGMN WVKQAPGKGLEWMGWINTYTGESTYADSFKGRFTFSLDTSASAAYLQINSLRAEDTAVYYCARFAIKGDY WGQGTLLTVSS SEQ ID NO: 120 VLDIQMTQSPSSLSASVGDRVTITCRSTKSLLHSNGI TYLYWYQQKPGKAPKLLIYQMSNLASGVPSRFSSSGSGTDFTLTISSLQPEDFATYYCAQNLEIPRTFGQ GTKVELK CEA SEQ ID NO: 121 HCDR1DTYMH SEQ ID NO: 122 HCDR2 RIDPANGNSKYADSVKG SEQ ID NO: 123 HCDR3FGYYVSDYAMAY SEQ ID NO: 124 LCDR1 RAGESVDIFGVGFLH SEQ ID NO: 125 LCDR2RASNLES SEQ ID NO: 126 LCDR3 QQTNEDPYT SEQ ID NO: 127 VHEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYMH WVRQAPGKGLEWVARIDPANGNSKYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAPFGYYVSD YAMAYWGQGTLVTVSS SEQ ID NO: 128 VLDIQLTQSPSSLSASVGDRVTITCRAGESVDIFGVG FLHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSGSRTDFTLTISSLQPEDFATYYCQQTNEDPYTFGQG TKVEIK CEA SEQ ID NO: 129 HCDR1TYWMS SEQ ID NO: 130 HCDR2 EIHPDSSTINYAPSLKD SEQ ID NO: 131 HCDR3LYFGFPWFAY SEQ ID NO: 132 LCDR1 KASQDVGTSVA SEQ ID NO: 133 LCDR2 WTSTRHTSEQ ID NO: 134 LCDR3 QQYSLYRS SEQ ID NO: 135 VHEVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMS WVRQAPGKGLEWIGEIHPDSSTINYAPSLKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPW FAYWGQGTPVTVSS SEQ ID NO: 136 VLDIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAW YQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEI K CEA SEQ ID NO: 137 HCDR1 EFGMNSEQ ID NO: 138 HCDR2 WINTKTGEATYVEEFKG SEQ ID NO: 139 HCDR3 WDFAYYVEAMDYSEQ ID NO: 140 LCDR1 KASAAVGTYVA SEQ ID NO: 141 LCDR2 SASYRKRSEQ ID NO: 142 LCDR3 HQYYTYPLFT SEQ ID NO: 143 VHQVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMN WVRQAPGQGLEWMGWINTKTGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARWDFAYYV EAMDYWGQGTTVTVSS SEQ ID NO: 144 VLDIQMTQSPSSLSASVGDRVTITCKASAAVGTYVAW YQQKPGKAPKLLIYSASYRKRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYYTYPLFTFGQGTKL EIK Her2 SEQ ID NO: 145 HCDR1 DTYIHSEQ ID NO: 146 HCDR2 RIYPTNGYTRYADSVKG SEQ ID NO: 147 HCDR3 WGGDGFYAMDYSEQ ID NO: 148 LCDR1 RASQDVNTAVA SEQ ID NO: 149 LCDR2 SASFLYSSEQ ID NO: 150 LCDR3 QQHYTTPPT SEQ ID NO: 151 VHEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY AMDYWGQGTLVTVSS SEQ ID NO: 152 VLDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE IK Her2 SEQ ID NO: 153 HCDR1 DYTMDSEQ ID NO: 154 HCDR2 DVNPNSGGSIYNQRFKG SEQ ID NO: 155 HCDR3 NLGPSFYFDYSEQ ID NO: 156 LCDR1 KASQDVSIGVA SEQ ID NO: 157 LCDR2 SASYRYTSEQ ID NO: 158 LCDR3 QQYYIYPYT SEQ ID NO: 159 VHEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMD WVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFY FDYWGQGTLVTVSS SEQ ID NO: 160 VLDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAW YQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVE IK Her2 SEQ ID NO: 161 HCDR1 DTYIHSEQ ID NO: 162 HCDR2 RIYPTNGYTRYDPKFQD SEQ ID NO: 163 HCDR3 WGGDGFYAMDYSEQ ID NO: 164 LCDR1 KASQDVNTAVA SEQ ID NO: 165 LCDR2 SASFRYTSEQ ID NO: 166 LCDR3 QQHYTTPPT SEQ ID NO: 167 VHQVQLQQSGPELVKPGASLKLSCTASGFNIKDTYIH WVKQRPEQGLEWIGRIYPTNGYTRYDPKFQDKATITADTSSNTAYLQVSRLTSEDTAVYYCSRWGGDGFY AMDYWGQGASVTVSS SEQ ID NO: 168 VLDIVMTQSHKFMSTSVGDRVSITCKASQDVNTAVAW YQQKPGHSPKLLIYSASFRYTGVPDRFTGSRSGTDFTFTISSVQAEDLAVYYCQQHYTTPPTFGGGTKVE IK EGFR SEQ ID NO: 169 HCDR1 NYGVHSEQ ID NO: 170 HCDR2 VIWSGGNTDYNTPFTS SEQ ID NO: 171 HCDR3 ALTYYDYEFAYSEQ ID NO: 172 LCDR1 RASQSIGTNIH SEQ ID NO: 173 LCDR2 YASESISSEQ ID NO: 174 LCDR3 QQNNNWPTT SEQ ID NO: 175 VHQVQLKQSGPGLVQPSQSLSITCTVSGF SLTNYGVH WVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYE FAYWGQGTLVTVSA SEQ ID NO: 176 VLDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHW YQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLE LK EGFR SEQ ID NO: 177 HCDR1 SGDYYWSSEQ ID NO: 178 HCDR2 YIYYSGSTDYNPSLKS SEQ ID NO: 179 HCDR3 VSIFGVGTFDYSEQ ID NO: 180 LCDR1 RASQSVSSYLA SEQ ID NO: 181 LCDR2 DASNRATSEQ ID NO: 182 LCDR3 HQYGSTP LT SEQ ID NO: 183 VHQVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYY WSWIRQPPGKGLEWIGYIYYSGSTDYNPSLKSRVTMSVDTSKNQFSLKVNSVTAADTAVYYCARVSIFGV GTFDYWGQGTLVTVSS SEQ ID NO: 184 VLEIVMTQSPATLSLSPGERATLSCRASQSVSSYLAW YQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQYGSTPLTFGGGTKAE IK EGFR SEQ ID NO: 185 HCDR1 NYYIYSEQ ID NO: 186 HCDR2 GINPTSGGSNFNEKFKT SEQ ID NO: 187 HCDR3QGLWFDSDGRGFDF SEQ ID NO: 188 LCDR1 RSSQNIVHSNGNTYLD SEQ ID NO: 189LCDR2 KVSNRFS SEQ ID NO: 190 LCDR3 FQYSHVPWT SEQ ID NO: 191 VHQVQLQQSGAEVKKPGSSVKVSCKASGYTFTNYYIY WVRQAPGQGLEWIGGINPTSGGSNFNEKFKTRVTITVDESTNTAYMELSSLRSEDTAFYFCARQGLWFDS DGRGFDFWGQGSTVTVSS SEQ ID NO: 192 VLDIQMTQSPSSLSASVGDRVTITCRSSQNIVHSNGN TYLDWYQQTPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCFQYSHVPWTFGQ GTKLQIT GPC-3 SEQ ID NO: 193 HCDR1DYEMH SEQ ID NO: 194 HCDR2 AIDPQTGNTAFNQKFKG SEQ ID NO: 195 HCDR3 FYSLTYSEQ ID NO: 196 LCDR1 RSSQSIVHSNGNTYLQ SEQ ID NO: 197 LCDR2 KVSNRFSSEQ ID NO: 198 LCDR3 FQGSHFPYA SEQ ID NO: 199 VHQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMH WVKQAPGQGLEWIGAIDPQTGNTAFNQKFKGRVTLTRDKSSSTVYMELSSLRSEDTAVYYCTRFYSLTYW GQGTLVTVSS SEQ ID NO: 200 VLDVLMTQSPLSLPVTLGQPASISCRSSQSIVHSNGN TYLQWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTYFTLKISRVEAEDVGVYYCFQGSHFPYAFGG GTKVEIK Mesothelin SEQ ID NO: 201HCDR1 IYGMH SEQ ID NO: 202 HCDR2 VIWYDGSHEYYADSVKG SEQ ID NO: 203 HCDR3DGDYYDSGSPLDY SEQ ID NO: 204 LCDR1 RASQSVSSYLA SEQ ID NO: 205 LCDR2DASNRAT SEQ ID NO: 206 LCDR3 QQRSNWPLT SEQ ID NO: 207 VHQVYLVESGGGVVQPGRSLRLSCAASGITFSIYGMH WVRQAPGKGLEWVAVIWYDGSHEYYADSVKGRFTISRDNSKNTLYLLMNSLRAEDTAVYYCARDGDYYDS GSPLDYWGQGTLVTVSS SEQ ID NO: 208 VLEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAW YQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVE IK Mucin1 SEQ ID NO: 209 HCDR1 SYVLHSEQ ID NO: 210 HCDR2 YINPYNDGTQYNEKFKG SEQ ID NO: 211 HCDR3 GFGGSYGFAYSEQ ID NO: 212 LCDR1 SASSSVSSSYLY SEQ ID NO: 213 LCDR2 STSNLASSEQ ID NO: 214 LCDR3 HQWNRYPYT SEQ ID NO: 215 VHQVQLQQSGAEVKKPGASVKVSCEASGYTFPSYVLH WVKQAPGQGLEWIGYINPYNDGTQYNEKFKGKATLTRDTSINTAYMELSRLRSDDTAVYYCARGFGGSYG FAYWGQGTLVTVSS SEQ ID NO: 216 VLDIQLTQSPSSLSASVGDRVTMTCSASSSVSSSYLY WYQQKPGKAPKLWIYSTSNLASGVPARFSGSGSGTDFTLTISSLQPEDSASYFCHQWNRYPYTFGGGTRL EIK Mucin1 SEQ ID NO: 217 HCDR1NYWMN SEQ ID NO: 218 HCDR2 EIRLKSNNYTTHYAESVKG SEQ ID NO: 219 HCDR3HYYFDY SEQ ID NO: 220 LCDR1 RSSKSLLHSNGITYFF SEQ ID NO: 221 LCDR2QMSNLAS SEQ ID NO: 222 LCDR3 AQNLELPPT SEQ ID NO: 223 VHEVQLVESGGGLVQPGGSMRLSCVASGFPFSNYWMN WVRQAPGKGLEWVGEIRLKSNNYTTHYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCTRHYYFD YWGQGTLVTVSS SEQ ID NO: 224 VLDIVMTQSPLSNPVTPGEPASISCRSSKSLLHSNGI TYFFWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCAQNLELPPTFGQ GTKVEIK CA125 SEQ ID NO: 225 HCDR1SYAMS SEQ ID NO: 226 HCDR2 TISSAGGYIFYSDSVQG SEQ ID NO: 227 HCDR3QGFGNYGDYYAMDY SEQ ID NO: 228 LCDR1 KSSQSLLNSRTRKNQLA SEQ ID NO: 229LCDR2 WASTRQS SEQ ID NO: 230 LCDR3 QQSYNLLT SEQ ID NO: 231 VHVKLQESGGGFVKPGGSLKVSCAASGFTFSSYAMSW VRLSPEMRLEWVATISSAGGYIFYSDSVQGRFTISRDNAKNTLHLQMGSLRSGDTAMYYCARQGFGNYGD YYAMDYWGQGTTVTVSS SEQ ID NO: 232 VLDIELTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTR KNQLAWYQQKPGQSPELLIYWASTRQSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQQSYNLLTFGP GTKLEVK BCMA SEQ ID NO: 233 HCDR1NYWMH SEQ ID NO: 234 HCDR2 ATYRGHSDTYYNQKFKG SEQ ID NO: 235 HCDR3GAIYDGYDVLDN SEQ ID NO: 236 LCDR1 SASQDISNYLN SEQ ID NO: 237 LCDR2YTSNLHS SEQ ID NO: 238 LCDR3 QQYRKLPWT SEQ ID NO: 239 VHQVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYWMH WVRQAPGQGLEWMGATYRGHSDTYYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGAIYDGY DVLDNWGQGTLVTVSS SEQ ID NO: 240 VLDIQMTQSPSSLSASVGDRVTITCSASQDISNYLNW YQQKPGKAPKLLIYYTSNLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRKLPWTFGQGTKLE IK

The anti-CD3 scFv binds to an effector cell at an EC₅₀ value greaterthan about 50 nM, or greater than 100 nM, or greater than 300 nM, orgreater than 500 nM in an in vitro FACS binding affinity assay; morepreferably, the second single-chain Fv of the bispecific antibody notonly binds to human CD3 but also specifically binds to CD3 of cynomolgusmonkeys or rhesus monkeys.

Some preferred amino acid sequences of the VH domain and itscomplementary determining regions (HCDR1, HCDR2, and HCDR3) and aminoacid sequences of the VL domain and its complementary determiningregions (LCDR1, LCDR2 and LCDR3) of the anti-CD3 scFv are exemplified inTable 6-2, wherein the amino acid residues contained in the CDRs aredefined according to the Kabat rule, wherein the amino acid compositionof the linker peptide between VH and VL of the anti-CD3 scFv is(GGGGS)_(n), wherein n=1, 2, 3, 4 or 5.

TABLE 6-2 Amino acid sequences of the anti-CD3 scFvincluded in the bispecific antibody andamino acid sequences of its CDR regions CD3-3 SEQ ID HCDR1 TYAMN NO: 241SEQ ID HCDR2 RIRSKYNNYATYYADSVKD NO: 242 SEQ ID HCDR3 HGNFGNSYVSWFAYNO: 243 SEQ ID LCDR1 RSSTGAVTTSNYAN NO: 244 SEQ ID LCDR2 GTNKRAP NO: 245SEQ ID LCDR3 ALWYSNLWV NO: 246 SEQ ID VHEVQLLESGGGLVQPGGSLKLSCAASGFTFNTYAMN NO: 247WVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID VL ELVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANO: 248 NWVQQKPGQAPRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNLWVFGGGTK LTVL CD3-4 SEQ ID HCDR1 KYAMNNO: 249 SEQ ID HCDR2 RIRSKYNNYATYYADSVKD NO: 250 SEQ ID HCDR3HGNFGNSYISYWAY NO: 251 SEQ ID LCDR1 GSSTGAVTSGYYPN NO: 252 SEQ ID LCDR2GTKFLAP NO: 253 SEQ ID LCDR3 ALWYSNRWV NO: 254 SEQ ID VHEVQLLESGGGLVQPGGSLKLSCAASGFTFNKYAMN NO: 255WVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRF TISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS SEQ ID VL ELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNO: 256 NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTK LTVL

The linker peptide that connects the anti-TAA scFv to the anti-CD3 scFvconsists of a flexible peptide and a rigid peptide; preferably, theamino acid composition of the flexible peptide has a general formulaG_(x)S_(y)(GGGGS)_(z), wherein x, y, and z are integers greater than orequal to 0, and x+y+z ≥1. The rigid peptide is derived from afull-length sequence (as shown in SEQ ID NO: 257) consisting of aminoacids at positions 118 to 145 of the carboxy terminus of the naturalhuman chorionic gonadotropin beta subunit, or a truncated fragmentthereof, preferably, the composition of the CTP rigid peptide isSSSSKAPPPS (CTP¹). Some preferred amino acid sequences of the linkerpeptide that connects the anti-TAA scFv and the anti-CD3 scFv areexemplified in Table 6-3.

TABLE 6-3 Amino acid sequences of the linker peptidethat connects the anti-TAA scFv and the anti-CD3 scFv SEQ ID NO:G₂(GGGGS)₃CTP¹ GGGGGGSGGGGSGGGGSSSSSK 258 APPPS SEQ ID NO: (GGGGS)₃CTP¹GGGGSGGGGSGGGGSSSSSKAP 259 PPS SEQ ID NO: GS(GGGGS)₂CTP¹GSGGGGSGGGGSSSSSKAPPPS 260 SEQ ID NO: (GGGGS)₁CTP⁴GGGGSSSSSKAPPPSLPSPSRL 261 PGPSDTPILPQ

The Fc fragment is directly connected or connected by a linker peptideto the anti-CD3 scFv, wherein the linker peptide includes 1 to 20 aminoacids that are preferably selected from the following amino acids:Gly(G), Ser(S), Ala(A), and Thr(T), more preferably Gly(G) and Ser(S);and most preferably, the linker peptide consists of (GGGGS)n, whereinn=1, 2, 3 or 4.

The Fc fragment is preferably selected from heavy chain constant regionsof human IgG1, IgG2, IgG3 and IgG4 and more particularly selected fromheavy chain constant regions of human IgG1 or IgG4; and Fc is mutated tomodify the properties of the bispecific antibody molecule, e.g., reducedaffinity to at least one of human FcγRs (FcγRJ, FcγRIIa or FcγRIIIa) andC1q, a reduced effector cell function, or a reduced complement function.In addition, the Fc fragment may also contain amino acid substitutionsthat change one or more other characteristics (e.g., an ability ofbinding to an FcRn receptor, the glycosylation of the antibody or thecharge heterogeneity of the antibody).

Some amino acid sequences of the Fc fragment with one or more amino acidmutations are exemplified in Table 6-4.

TABLE 6-4 Amino acid sequences of Fc from human IgGAmino acid sequence of a constant region of anIgG1 Fc (L234A/L235A) mutant (EU numbering) SEQ IDDKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT NO: 262CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTYRVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAKGQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVMHE ALHNHYTQKS LSLSPGKAmino acid sequence of a constant region of anIgG1 (L234A/L235A/T250Q/N297A/ P331S/M428L/K447-)mutant (EU numbering)SEQ ID DKTHTCPPCP APEAAGGPSV FLFPPKPKDQ LMISRTPEVT NO: 263CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYASTYRVVSVLTVLH QDWLNGKEYK CKVSNKALPA SIEKTISKAKGQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVEWESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQGNVFSCSVLHE ALHNHYTQKS LSLSPGK

Amino acid sequences of some preferred bispecific antibodies andcorresponding nucleotide sequences thereof are exemplified in Table 6-5.

TABLE 6-5 several bispecific antibodies of scFv1-scFv2-Fc configurationAntibody Amino acid Nucleotide sequence code Target site sequence No.No. AB1K1 Anti-CD19 × CD3 SEQ ID NO: 264 SEQ ID NO: 265 AB1K2 Anti-CD19× CD3 SEQ ID NO: 283 SEQ ID NO: 284 AB2K Anti-CD20 × CD3 SEQ ID NO: 266SEQ ID NO: 267 AB3K Anti-CD22 × CD3 SEQ ID NO: 268 SEQ ID NO: 269 AB4KAnti-CD30 × CD3 SEQ ID NO: 270 SEQ ID NO: 271 AB5K Anti-EpCAM × CD3  SEQID NO: 272 SEQ ID NO: 273 AB6K  Anti-CEA × CD3 SEQ ID NO: 274 SEQ ID NO:275 AB7K7  Anti-Her2 × CD3 SEQ ID NO: 8 SEQ ID NO: 276 AB8K Anti-EGFR ×CD3  SEQ ID NO: 277 SEQ ID NO: 278 AB9K Anti-GPC-3 × CD3  SEQ ID NO: 279SEQ ID NO: 280 AB10K Anti-Mesothelin × CD3    SEQ ID NO: 281 SEQ ID NO:282 AB11k Anti-Mucin 1 × CD3  SEQ ID NO: 285 SEQ ID NO: 286

Example 7 Pharmacodynamics Study of Anti-GPC-3×CD3 Bispecific Antibodiesin a Mouse Transplanted Tumor Model 7.1 NOD-SCID Mouse Model ofTransplanted Tumor Constructed by Subcutaneously Co-Inoculating HumanPBMCs and Human Liver Cancer Cells Huh-7

GPC-3-positive human liver cancer cells Huh-7 were selected to study theinhibiting effect of bispecific antibodies on tumor growth in vivo in aNOD-SCID mouse model of transplanted tumor constructed by subcutaneouslyco-inoculating human PBMC cells and Huh-7 cells.

The peripheral blood of a normal human was subjected to density gradientcentrifugation to separate human PBMCs. Female NOD-SCID mice (purchasedfrom Shanghai Lingchang Biotechnology Co., Ltd.) at the age of seven toeight weeks were selected and Huh-7 cells in the logarithmic growthstage were collected. 3×10⁶ Huh-7 cells and 3×10⁶ PBMCs were mixed andinoculated subcutaneously on the right back of each NOD-SCID mouse. Onehour after inoculation, the mice were randomly divided into two groupswith six mice in each group according to their weights. The treatedgroup was intraperitoneally administered with AB9K at a dose of 1 mg/kg,and the control group was administered with a PBS solution of the samevolume, once a day continuously for 6 days. The day of administrationwas recorded as Day 0. The maximum diameter (D) and the minimum diameter(d) of the tumor were measured weekly. The volume (mm³) of the tumor ofeach group and the tumor growth inhibition rate (TGI) (%) of eachtreated group were calculated using the formulas as shown in Example3.1.

As shown in FIG. 6-1, on Day 21 of administration, the average tumorvolume of the PBS control group was 1311.03±144.89 mm³; the averagetumor volume of the treated group administrated with AB9K was60.83±12.63 mm³, and the TGI was 95.36%, wherein the tumor in one mousewas completely regressed, which was significantly different from that ofthe control group (P<0.001). The above results show that most of PBMCsare inactivated primary T cells, the bispecific antibody AB9K canactivate the primary T cells in the animals and draw the distancebetween the T cells and the target cell Huh-7 so that the T cells candirectly kill the tumor cells and inhibit the growth of the tumor, andthus AB9K has a very good anti-tumor effect at a dose of 1 mg/kg.

7.2 NPG Mouse Model of Transplanted Tumor Constructed by SubcutaneouslyCo-Inoculating Human CIK Cells and Human Burkkit's Lymphoma Raji Cells

GPC-3-negative human Burkkit's lymphoma Raji cells were selected tostudy the inhibiting effect of bispecific antibodies on tumor growth invivo in an NPG mouse model of transplanted tumor constructed bysubcutaneously co-inoculating human CIK cells and human Burkkit'slymphoma Raji cells.

CIK cells were prepared in the method as described in Example 3.1.Female NPG mice at the age of seven to eight weeks were selected, andRaji cells in the logarithmic growth stage were collected. 5×10⁶ Rajicells and 2×10⁶ CIK cells were mixed and inoculated subcutaneously onthe right back of each NPG mouse. One hour after inoculation, the micewere randomly divided into three groups with five mice in each groupaccording to their weights. The treated group was intraperitoneallyadministered with AB9K at a dose of 1 mg/kg, and the control group wasadministered with a PBS solution of the same volume, once a daycontinuously for 10 days. The day of administration was recorded as Day0. The maximum diameter (D) and the minimum diameter (d) of the tumorwere measured weekly. The volume (mm³) of the tumor of each group andthe tumor growth inhibition rate (TGI) (%) of each treated group werecalculated using the formulas as shown in Example 3.1.

As shown in FIG. 6-2, on Day 26 of administration, the average tumorvolume of the PBS control group was 2636.66±196.62 mm³; the averagetumor volume of the treated group administrated with AB9K was2739.57±220.13 mm³, which was not significantly different from that ofthe control group. In summary, the results show that bispecific antibodyAB9K exhibited no non-specific killing on GPC-3-negative cell strains,which indicates that the bispecific antibody does not mediate T cells tokill non-target tissues in vivo, there is no drug toxicity, and thesafety is high.

7.3 CD34 Immune-Reconstituted NPG Mouse Model of Transplanted TumorConstructed by Inoculating Human Liver Cancer Huh-7 Cells

GPC-3-positive human liver cancer Huh-7 cells were selected to study theinhibiting effect of bispecific antibodies on tumor growth in vivo in aCD34 immune-reconstituted NPG mouse model of transplanted tumorconstructed by subcutaneously inoculating human liver cancer Huh-7cells.

CD34 immune-reconstituted NPG mice were prepared in the method asdescribed in Example 3.5. Huh-7 cells in the logarithmic growth stagewere collected and 2.5×10⁶ Huh-7 cells were inoculated subcutaneously onthe right back of the immune-reconstituted mice. Four days afterinoculation, the mice were randomly divided into two groups with sevenmice in each group according to the tumor volumes and weights. Thetreated group was intraperitoneally administered with AB9K at a dose of1 mg/kg, and the control group was administered with a PBS solution ofthe same volume, once a day until the test was completed. The day ofadministration was recorded as Day 0. The maximum diameter (D) and theminimum diameter (d) of the tumor were measured weekly. The volume (mm³)of the tumor of each group and the tumor growth inhibition rate (TGI)(%) of each treated group were calculated using the formulas as shown inExample 3.1.

As shown in FIG. 6-3, on Day 21 of administration, the average tumorvolume of the PBS control group was 2102.84±275.71 mm³; the averagetumor volume of the treated group administrated with AB9K at the dose of1 mg/kg was 325.01±282.21 mm³, and the TGI was 86.53%, wherein the tumorin four mice was completely regressed, which was significantly differentfrom that of the control group (P<0.001). The above results show thatthe bispecific antibody AB9K had an excellent anti-tumor effect in theCD34 immune-reconstituted model.

Example 8 In Vitro Biological Function Evaluation of Anti-CD20×CD3Bispecific Antibodies and Pharmacodynamics Study of Anti-CD20×CD3Bispecific Antibodies in a Mouse Transplanted Tumor Model 8.1 Detectionof the Binding Activity of AB2K to CD20-Positive Tumor Cells by FlowCytometry

Raji cells (purchased from the cell bank of Chinese Academy of Sciences)were cultured and collected by centrifugation. The collected cells wereresuspended with 1% PBSB and placed in 96-well plates, 100 μl (i.e.,2×10⁵ cells) per well, after the cell density was adjusted to (2×10⁶)cells/ml. Diluted bispecific antibodies with a series of concentrationswere added and incubated for 1 hour at 4° C. The cells were centrifugedto discard the supernatant and then washed three times using a PBSsolution with 1% BSA (PBSB). Diluted AF488-labeled goat anti-human IgGantibodies (Jackson Immuno Research Inc., Cat. No. 109-545-088) or mouseanti-6×His IgG antibodies (R&D Systems, Cat. No. IC050P) were added tothe cells, and the cells were incubated for 1 hour at 4° C. in the dark.The obtained cells were centrifuged to discard the supernatant and thenwashed twice with 1% PBSB, and cells in each well were resuspended with100 μl of 1% paraformaldehyde. The signal intensity was detected by flowcytometry. The analysis was performed with the average fluorescenceintensity as the Y-axis and the antibody concentration as the X-axisthrough software GraphPad to calculate the EC₅₀ value for the binding ofAB2K to Raji cells.

As shown in FIG. 7-1, AB2K bound well to CD20-positive cells, the signalintensity was proportional to the antibody concentration, and the EC₅₀value for AB2K binding to Raji cells was calculated, which was about69.97 nM.

8.2 AB2K Mediating Effector Cells to Target and Kill CD20-Positive TumorCells

Normally cultured Raji-luc cells (purchased from Beijing BiocytogenBiotechnology Co., Ltd.) were added to 96-well white plates after thecell density was adjusted to 1×10⁵ cells/ml, 40 μl per well. AB2Kantibodies were diluted into a series of gradients and added to the96-well white plates. After the CIK cell density was adjusted to 5×10⁵cells/ml, the CIK cells were added to the 96-well white plates, 40 μlper well, to make the effector:target ratio (E:T) equal to 5:1, andcultured for 24 hours at 37° C. After 24 hours, the white plates weretaken out, 100 μl of One-Glo (Promega, Cat. No. E6120) solution wasadded to each well, and then the white plates were placed for at leastthree minutes at room temperature. The luminescence value was measuredby a microplate reader. The analysis was performed with the fluorescenceintensity as the Y-axis and the antibody concentration as the X-axisthrough software GraphPad to calculate the EC₅₀ value of AB2K killingRaji-luc cells.

As shown in FIG. 7-2, the EC₅₀ for AB2K mediating effector cells to killRaji-luc cells was only 42.8 ng/ml and AB2K had target specificity,while the EC₅₀ of AB7K7 as a negative control was 229.5 ng/ml and AB7K7had little killing effect on Raji-luc cells.

8.3 Evaluation of Abilities of Bispecific Antibodies to Activate T CellsThrough Reporter Gene Cell Strains

Jurkat T cells containing NFAT RE reporter genes (BPS Bioscience, Cat.No. 60621) can overexpress luciferase in the presence of bispecificantibodies and CD20-positive Raji cells, and the degree of activation ofthe Jurkat T cells can be quantified by detecting the activity of theluciferase. A four-parameter curve was fitted using the concentration ofbispecific antibody as the X-axis and the fluorescein signal as theY-axis.

As shown in FIG. 7-3, AB2K can specifically activate Jurkat NFATRE Luccells, wherein the EC₅₀ value was 0.2006 μg/ml and its concentration wasproportional to signal intensity, while AB7K7 as a negative control hadlittle ability to activate T cells.

8.4 NPG Mouse Model of Transplanted Tumor Constructed by SubcutaneouslyCo-Inoculating Human CIK Cells and Human Burkkit's Lymphoma Raji Cells

CD20-positive human Burkkit's lymphoma Raji cells were selected to studythe inhibiting effect of bispecific antibodies on tumor growth in vivoin an NPG mouse model of transplanted tumor constructed bysubcutaneously co-inoculating human CIK cells and human Burkkit'slymphoma cells Raji.

CIK cells were prepared in the method as described in Example 3.1.Female NPG mice (purchased from Beijing Vitalstar Biotechnology Co.,Ltd.) at the age of seven to eight weeks were selected, and Raji cellsin the logarithmic growth stage were collected. 4×10⁶ Raji cells and8×10⁵ CIK cells were mixed and inoculated subcutaneously on the rightback of each NPG mouse. One hour after inoculation, the mice wererandomly divided into five groups with six mice in each group accordingto their weights and intraperitoneally administered with correspondingdrugs. Specifically, all treated groups were administered with Rituxan(from Roche) and bispecific antibody AB2K, respectively, at doses of 1mg/kg and 0.1 mg/kg, twice a week. The day of administration wasrecorded as Day 0. The maximum diameter (D) and the minimum diameter (d)of the tumor were measured weekly. The volume (mm³) of the tumor of eachgroup and the tumor growth inhibition rate (TGI) (%) of each treatedgroup were calculated using the formulas as shown in Example 3.1.

As shown in FIG. 7-4, on Day 24 of administration, the average tumorvolume of the PBS control group was 1766.84±155.62 mm³; the averagetumor volume of the treated group administrated with Rituxan at a doseof 1 mg/kg was 647.92±277.11 mm³, and TGI was 63.33%, which wassignificantly different from that of the control group (P<0.01); theaverage tumor volume of the treated group administrated with Rituxan ata dose of 0.1 mg/kg was 1893.81±186.99 mm³, and Rituxan herein exhibitedno efficacy; the average tumor volume of the treated group administratedwith AB2K at a dose of 1 mg/kg was 116.18±39.50 mm³, and TGI was 93.42%,which was significantly different from that of the control group(P<0.01); the average tumor volume of the treated group administratedwith AB2K at a dose of 0.1 mg/kg was 1226.03±340.05 mm³, and TGI was30.61%, which was not significantly different from that of the controlgroup. The results show that the bispecific antibody AB2K could inhibitthe growth of tumor cells by activating human immune cells in animals;and at the same dose, the efficacy of the bispecific antibody was betterthan the efficacy of the monoclonal antibody Rituxan, and the bispecificantibody exhibited great anti-tumor effects.

8.5 NPG Mouse Model of Transplanted Tumor Constructed by SubcutaneouslyCo-Inoculating Human CIK Cells and Human Burkkit's Lymphoma Daudi Cells

CD20-positive human Burkkit's lymphoma Daudi cells were selected tostudy the inhibiting effect of bispecific antibodies on tumor growth invivo in an NPG mouse model of transplanted tumor constructed bysubcutaneously co-inoculating human CIK cells and human Burkkit'slymphoma Daudi cells.

CIK cells were prepared in the method as described in Example 3.1.Female NPG mice at the age of seven to eight weeks were selected, andDaudi cells in the logarithmic growth stage were collected. 4×10⁶ Daudicells and 8×10⁵ CIK cells were mixed and inoculated subcutaneously onthe right back of each NPG mouse. One hour later, the mice were randomlydivided into five groups with six mice in each group according to theirweights and intraperitoneally administered with corresponding drugs. Alltreated groups were administrated twice a week. Rituxan and bispecificantibody AB2K were both administered at doses of 1 mg/kg and 0.1 mg/kg,respectively. The day of administration was recorded as Day 0. Themaximum diameter (D) and the minimum diameter (d) of the tumor weremeasured weekly. The volume (mm³) of the tumor of each group and thetumor growth inhibition rate (TGI) (%) of each treated group werecalculated using the formulas as shown in Example 3.1.

As shown in FIG. 7-5, on Day 30 of administration, the average tumorvolume of the PBS control group was 889.68±192.13 mm³; the average tumorvolume of the treated group administrated with Rituxan at a dose of 1mg/kg was 241.51±44.91 mm³, and TGI was 72.85%, which was significantlydifferent from that of the control group (P<0.01); the average tumorvolume of the treated group administrated with Rituxan at a dose of 0.1mg/kg was 746.11±299.71 mm³, which was not significantly different fromthat of the control group; the average tumor volume of the treated groupadministrated with AB2K at a dose of 1 mg/kg was 72.05±11.89 mm³, andTGI was 91.9%, which was significantly different from that of thecontrol group (P<0.01); the average tumor volume of the treated groupadministrated with AB2K at a dose of 0.1 mg/kg was 75.36±11.81 mm³, andTGI was 91.53%, which was significantly different from that of thecontrol group (P<0.01). The results show that the bispecific antibodyAB2K could inhibit the growth of tumor cells by activating human immunecells in animals; and at the same dose, the efficacy of the bispecificantibody was better than the efficacy of the monoclonal antibodyRituxan, and AB2K exhibited good anti-tumor effects even at a low dose.

Example 9 Evaluation of the Safety of Anti-CD20×CD3 BispecificAntibodies

The toxicity of AB2K was evaluated to determine appropriate dose rangesand observation indicators for subsequent toxicity tests. Adult Femalecynomolgus monkeys (purchased from Guangzhou Xiangguan BiotechnologyCo., Ltd.) at the age of 3-4 years and with the weight of 3-4 kg weredivided into two groups with one mouse in each group, wherein the twogroups were a vehicle control group and an AB2K treated group. Thegroups were administrated via intravenous drip by a peristaltic pump for1 hour. The dose amount and volume administered are shown in Table 7.The groups were administrated on Day 0 (D0), Day 7 (D7), Day 21 (D21),and Day 28 (D28), respectively, for a total of four doses, and the drugdose was gradually escalated each time. The monkeys were weighed weekly.

TABLE 7 Dosing schedule for cynomolgus monkey acute toxicity evaluationTo-be-tested Group drugs name Dose volume Dose amount G1 Vehicle D0: 5mL/kg N/A control D7: 5 mL/kg group D21: 10 mL/kg D28: 10 mL/kg G2 AB2KD0: 5 mL/kg D0: 0.06 mg/kg D7: 5 mL/kg D7: 0.3 mg/kg D21: 10 mL/kg D21:1.5 mg/kg D28: 10 mL/kg D28: 3 mg/kg

During the test, animals were periodically monitored for clinicalsymptoms, body weight, food consumption, body temperature,electrocardiogram, blood pressure, clinicopathological indexes (bloodcell count, coagulation function measure, and blood biochemistry),lymphocyte subsets, cytokines, drug plasma concentration measure, andtoxicokinetics analyses. After administration of AB2K, the physicalsigns of cynomolgus monkeys exhibited no abnormal reaction, the bodyweight was relatively stable, the body temperature fluctuation wassimilar to the body temperature fluctuation of the vehicle controlgroup, and no death or impending death was observed among animals duringthe administration period. As shown in FIG. 8, after administration, thewhite blood cell changes of cynomolgus monkeys in the AB2K group weresimilar to the white blood cell changes in the control group; the firstadministration of AB2K at a dose of 0.06 mg/kg had little effect onlymphocytes; 1 hour to 6 hours after the second administration, thenumber of lymphocytes in the animals of the treated group decreasedsharply and recovered to normal after 24 hours; as the number ofadministrations increased, the effect of AB2K on the decrease in thenumber of lymphocytes was weaker and weaker despite increasing doses. Inaddition, after the first administration of AB2K, the release of IL-2,IL-6 and TNF-α factors was promoted and the release of IL-5 was slightlystimulated, but the release of IFN-γ was not stimulated; as the numberof administrations increased, the release-promoting effect of AB2K oncytokines became less and less significant, indicating that the body hadalready been adapted to the stimulation by bispecific antibodies.

Example 10 Pharmacokinetics Evaluation of Anti-CD20×CD3 BispecificAntibodies

Female cynomolgus monkeys with the weight of 3-4 kg were divided intotwo groups with one in one monkey in each group. The first group was ablank control group, and the second group was an AB2K treated groupadministrated at a dose of 0.3 mg/kg. The blood sampling time pointswere Minute 15, Hour 1, Hour 3, Hour 6, Hour 10, Hour 24, Hour 30, Hour48, Hour 54, Hour 72, Hour 96, and Hour 144, respectively, a total of 13time points. Serum was collected from blood and frozen at −80° C.

The drug concentration of AB2K in serum was determined by ELISA. Thepharmacokinetics parameters were calculated using software PKSolver.Specific parameters are shown in Table 8. The results show that T_(1/2)of AB2K in normal cynomolgus monkeys was about 8.5 hours.

TABLE 8 Pharmacokinetics parameters of bispecific antibody AB2K incynomolgus monkeys AUC 0-inf_obs Vz_obs Cl_obs AB2K t_(1/2) (h)(μg/mL*h) (μg/kg)/(μg/mL) (μg/kg)/(μg/mL)/h Pharmacokinetics 8.45 168.6321.68 1.78 parameter

Example 11 Evaluation of In Vitro Biological Functions of Anti-CD19×CD3Bispecific Antibodies 11.1 Detection of Binding Activities of BispecificAntibodies to Effector Cells and Target Cells (FACS) a) Detection ofBinding Activities of Bispecific Antibodies to CD19-Positive Tumor RajiCells by Flow Cytometry

CD19-positive tumor cells Raji cells were cultured and collected bycentrifugation. The collected cells were resuspended with 1% PBSB,placed in 96-well plates after the cell density was adjusted to (2×10⁶)cells/ml, 100 μl (2×10⁵ cells) per well, and blocked for 0.5 hours at 4°C. The blocked cells were centrifuged to discard the supernatant, andthen diluted bispecific antibodies AB1K2 with a series of concentrationsand isotype CD19 bispecific antibodies AB23P8, AB23P9 and AB23P10 wereadded and incubated for 1 hour at 4° C. The cells were centrifuged todiscard the supernatant and then washed three times using PBSB with 1%BSA. Diluted AF647-labeled goat anti-human IgG antibodies were added tothe cells, and the cells were incubated for 1 hour at 4° C. in the dark.The obtained cells were centrifuged to discard the supernatant andwashed twice with 1% PBSB, and cells in each well were resuspended with100 μl of 1% PF. The signal intensity was detected by flow cytometry.The analysis was performed with the average fluorescence intensity asthe Y-axis and the antibody concentration as the X-axis through softwareGraphPad to calculate the EC₅₀ value for the binding of bispecificantibodies to tumor cells Raji.

The results show that bispecific antibodies with different structureshad a good binding activity to tumor cells over-expressing CD19. FIG.9-1 shows binding curves of bispecific antibodies with differentstructures to tumor cells Raji. As shown in Table 9-1, EC₅₀ for thebinding of each of four bispecific antibodies to tumor cells Raji was atthe nM level.

TABLE 9-1 Detection of abilities of Anti-CD19 × CD3 bispecificantibodies to bind to tumor cells Raji AB1K2 AB23P8 AB23P9 AB23P10 EC₅₀(nM) 1.393 1.924 2.600 2.678

b) Detection of Binding Activities of Bispecific Antibodies to Human TCells by FACS

PBMCs were prepared from fresh human blood by density gradientcentrifugation. The prepared PBMCs were resuspended in a 1640 mediumcontaining 10% heat-inactivated FBS, added with 2 μg/ml of CD3 antibodyfor activation for 24 h, then added with 250 IU/ml of IL-2 foramplification for 7 days, to prepare expanded T cells which weredetected by flow cytometry to be positive for CD3 expression on thesurface. The to-be-detected sample was prepared and detected in the samemanner as in a) of Example 11.1. Cells resuspended with 1% PF weredetected on a machine and, with the average fluorescence intensity,analyzed by software GraphPad to calculate EC₅₀ value for the binding ofeach bispecific antibody to human T cells.

The results in FIG. 9-2 show that each bispecific antibody had a goodbinding activity to CIK. As shown in Table 9-2, the EC₅₀ of AB1K2 wasabout 16 nM, which was roughly equal to the EC₅₀ of AB23P8, and the EC₅₀of AB23P9 and AB23P10 were about 50 nM and 30 nM, respectively.

TABLE 9-2 Detection of abilities of Anti-CD19 × CD3 bispecificantibodies to bind to effector cells CIK AB1K2 AB23P8 AB23P9 AB23P10EC₅₀ (nM) 15.69 16.69 49.52 32.41c) Detection of Cross-Reactivity of Bispecific Antibodies with CD3 onthe Surface of Cynomolgus Monkey CIK Cell Membrane by FACS

PBMCs were prepared from fresh cynomolgus monkey blood by densitygradient centrifugation. The prepared PBMCs were resuspended in a 1640medium containing 10% heat-inactivated FBS, added with 2 μg/ml of OKT3for activation for 24 h, then added with 250 IU/ml of IL-2 foramplification for 7 days to prepare cynomolgus monkey CIK cells for use.Human CIK cells and cynomolgus monkey CIK cells were collected bycentrifugation. The to-be-detected sample was prepared and detected inthe same manner as in a) of Example 11.1. Cells resuspended with 1%paraformaldehyde solution were detected on a machine and, with theaverage fluorescence intensity, analyzed by software GraphPad tocalculate the EC₅₀ values for the binding of bispecific antibodies tohuman CIK cells and the EC₅₀ values for the binding of bispecificantibodies to cynomolgus monkey CIK cells.

As shown in FIG. 9-3, there was no difference between the bindingability of the bispecific antibody AB1K2 to cynomolgus monkey T cellsand the ability of the bispecific antibody AB23P10 to cynomolgus monkeyT cells, the EC₅₀ for the binding of each of both bispecific antibodiesto cynomolgus monkey T cells was approximately 5.5 nM as detected byflow cytometry, and the ability of the both bispecific antibodies tocynomolgus monkey T cells was stronger than the ability of the bothbispecific antibodies to human T cells.

11.2 Detection of Abilities of Bispecific Antibodies to Bind to Antigens

The binding of bispecific antibodies to soluble CD3 and CD19 wasdetected by double antigen sandwich ELISA.

CD19 proteins (ACRO Biosystems, Cat. No. CD9-H5251) were diluted withPBS to a concentration of 1 μg/ml and added to 96-well plates, 100 μlper well. The plates were coated at 4° C. overnight. The plates werethen blocked with 1% skimmed milk powder for 1 hour at room temperature.Each bispecific antibody was diluted with a 5-fold gradient for a totalof 10 concentration gradients. The 96-well plates were then washed withPBST, and then the diluted bispecific antibodies were added. Controlwells without antibodies were set. Incubated for 2 hours at roomtemperature. Unbound bispecific antibodies were washed away with PBST.Biotinylated CD3E&CD3D (ACRO Biosystem, Cat. No. CDD-H82W1) were mixedat 50 ng/ml with streptavdin HRP (BD, Cat. No. 554066), added in 96-wellplates, 100 μl per well, and incubated for 1 hour at room temperature.96-well plates were washed with PBST, and TMB was added to the plates,100 μl per wells. Color development was performed at room temperaturefor 15 minutes, and then 0.2 M H₂SO₄ was added to stop the colordevelopment reaction. The light absorbance values at A450-620 nm weremeasured by a microplate reader. Analysis was performed by softwareGraphPa, and the EC₅₀ values for the binding of bispecific antibodies totwo antigens were calculated.

The results show that each bispecific antibody bound specifically toboth CD3 and CD19 molecules and exhibited good dose-dependence as theconcentration of the antibodies changed (FIG. 9-4). The abilities ofseveral bispecific antibodies to bind to soluble CD3 and CD19 are shownin Table 9-3, with EC₅₀ values ranging from 0.19 nM to 0.47 nM, andthere was little difference between binding activities at both ends.

TABLE 9-3 Detection of abilities of Anti-CD19 × CD3 bispecificantibodies to bind to CD3 and CD19 molecules AB1K2 AB23P8 AB23P9 AB23P10EC₅₀ (nM) 0.2185 0.1925 0.2211 0.4704

11.3 Evaluation of Abilities of Bispecific Antibodies to Activate TCells Through Reporter Gene Cell Strains

Jurkat T cells containing NFAT RE reporter genes can overexpressluciferase in the presence of bispecific antibodies and target cellsRaji, and the degree of activation of the Jurkat T cells can bequantified by detecting the activity of the luciferase. A four-parametercurve was fitted using the concentration of bispecific antibody as theX-axis and the fluorescein signal as the Y-axis.

The test results in FIGS. 9-5 and 9-6 show that Jurkat T cells canhardly be activated in the absence of target cells overexpressing CD19,and T cells can be activated only in the presence of both the bispecificantibody and the target cells at both ends. The ability of eachbispecific antibody to activate Jurkat T cells is shown in Table 9-4,and the ability of each bispecific antibody to activate Jurkat T cellswas almost equivalent to each other.

TABLE 9-4 Detection of abilities of Anti-CD19 × CD3 bispecificantibodies to activate a reporter gene cell strain that are Jurkat Tcells AB1K2 AB23P8 AB23P9 AB23P10 Blincyto EC₅₀ (nM) 1.080 1.123 0.85270.7093 2.714

11.4 Abilities of Bispecific Antibodies to Mediate T Cells to Kill TumorCells

Normally cultured tumor cell lines, including Raji-Luc, NALM6 and Rehcells (all purchased from the cell bank of Chinese Academy of Sciences,Shanghai) were used as target cells, and cell suspensions were collectedand centrifuged, added to 96-well cell culture plates after the celldensity was adjusted to 2×10⁵ cells/ml, 100 μl per well, and culturedovernight. The antibodies were diluted according to the test design, andadded to the cells, 50 μl per well, while wells without the addition ofantibodies were supplemented with the same volume of the medium.Effector cells (human PBMCs or expanded CIK cells) whose number was fivetimes larger than the number of target cells, were then added, 100 μlper well. Control wells were set, and wells without the addition ofeffector cells were supplemented with the same volume of the medium.After 48 hours of culture, Raji-Luc cells were detected by Steady-GloLuciferase Assay System (Promega) and other cells were detected byCytoTox96 Non-Radio Cytotoxicity Assay (Promega). The analysis wasperformed with the detection results as the Y-axis and the bispecificantibody concentration as the X-axis through software GraphPad tocalculate and compare the ability of each bispecific antibody to mediatethe killing on Raji-luc cells.

The EC₅₀ values of each bispecific antibody to mediate effector cells tokill tumor cells are shown in Tables 9-5 to 9-7. The results show thateach bispecific antibody exhibited a very significant killing effect ontumor cells with high expression of CD19 in a dose-dependent manner,wherein EC₅₀ of each bispecific antibody reached the pM level.

TABLE 9-5 EC₅₀ values of bispecific antibodies to mediate CIK to killtumor cells EC₅₀ (pM) AB1K2 AB23P8 AB23P9 AB23P10 Blincyto Raji-LUC0.6988 0.5861 0.1480 0.1280 0.5952 0.2024 — — 0.4834 5.654 Note: —meansthat no detection is performed.

TABLE 9-6 EC₅₀ values of bispecific antibodies to mediate PBMCs to killtumor cells EC₅₀ (pM) AB1K2 AB23P8 AB23P9 AB23P10 Blincyto Raji-LUC1.225 1.025 1.014 0.9462 5.452 1.254 — — 1.254 21.22 — — — 4.176 22.58Note: —means that no detection is performed.

TABLE 9-7 EC₅₀ values of bispecific antibodies to mediate CIK to killdifferent tumor cells EC₅₀ (pM) AB1K2 AB23P10 Blincyto NALM6 — 4.40277.29 Reh 1.709 1.640 11.87 Note: —means that no detection is performed.

Example 12 Evaluation of In Vitro Biological Functions ofAnti-Mucin1×CD3 Bispecific Antibodies 12.1 Binding Activities of AB11Kto Tumor Cells Over-Expressing Mucin1 and to Human or Cynomolgus MonkeyPrimary T Cells

Human breast cancer cells MCF-7, BT-549, HCC70, T-47D and HCC1954, humanovarian cancer cell SK-OV-3, human cervical cancer cell Hela and humancolon cancer cell HT-29 were cultured, wherein MCF-7, BT-549, T-47D,HCC1954, SK-OV-3, Hela and HT-29 cells were purchase from the cell bankof Chinese Academy of Sciences, and HCC70 cells were purchase fromNanjing Cobioer Biotechnology Co., Ltd. Each kind of the above cells wasdigested with trypsin, collected by centrifugation, resuspended with 1%PBSB, placed in 96-well plates after the cell density of each kind ofcells was adjusted to 5×10⁵ cells/ml, 100 μl per well, and blocked for30 minutes at 4° C. Human or cynomolgus monkey primary T cells werecollected by centrifugation, resuspended with 1% PBSB, placed in 96-wellplates after the cell density of each kind of cells was adjusted to5×10⁵ cells/ml, 100 μl per well, and blocked for 30 minutes at 4° C. Thecells were washed once with 1% PBSB. Diluted AB11K with a series ofconcentrations was added at 100 μl per well and incubated for 1 hour at4° C. The cells were centrifuged to discard the supernatant and thenwashed twice with 1% PBSB. Diluted AF647 goat anti human IgG (H+L)antibodies (Jackson Immuno Research Inc., diluted at 1:250) were added,100 μl per well, and then the cells were incubated for 1 hour at 4° C.in the dark. The cells were centrifuged to discard the supernatant. Theplates were washed, and after that, 4% PFA was added, 150 μl per well,to resuspend the cells. The signal intensity was detected by flowcytometry. The analysis was performed with the average fluorescenceintensity as the Y-axis and the antibody molar concentration as theX-axis through software GraphPad Prism 6 to calculate the EC₅₀ valuesfor the binding of AB11K to the above tumor cells and human orcynomolgus monkey primary T cells.

As shown in FIG. 10-1 and Table 10-1, at the cellular level, AB11K boundto the above tumor cells and human or cynomolgus monkey primary T cells,and the signal intensity was proportional to the antibody concentration,and EC₅₀ calculated for the binding of AB11K to the above tumor cellsranged from 5 nM to 300 nM, wherein the binding to T-47D and Hela wasstrongest, followed by the binding to HCC70, HCC1954, SKOV-3 and BT-549,while the binding to MCF-7 and HT-29 was the weakest, which did notreach the upper platform. The EC₅₀ values for the binding of AB7K tohuman or cynomolgus monkey T cells were 13.43 nM and 9.996 nM,respectively, and the ability of AB11K to bind to cynomolgus monkey Tcells was roughly equivalent to the ability of AB11K to bind to human Tcells.

TABLE 10-1 EC₅₀ results for the binding of AB11K to tumor cellsover-expressing Mucin 1 and to human or cynomolgus monkey primary Tcells Cell name EC₅₀ (nM) MCF-7 / BT-549 287.2 HCC70 58.98 T-47D 5.053HCC1954 81.24 Hela 5.515 SK-OV-3 93.72 HT-29 / Human T cells 13.43cynomolgus monkey 9.996 T cells

12.2 Ability of AB11K to Mediate T Cells to Kill Tumor Cells

Normally cultured cells MCF-7, BT-549, HCC70, T-47D, HCC1954, SK-OV-3,Hela and HT-29 were used as target cells, respectively. Each kind ofcells was digested with trypsin, placed in 96-well cell culture platesafter the cell density of each kind of cells was adjusted to 2×10⁵cells/ml, 100 μl per well, and cultured overnight at 37° C. with 5% CO₂.Effector cells (expanded T cells) whose number was five times largerthan the number of corresponding target cells were added as T cellgroup, and effector cells (PBMCs from healthy volunteers) whose numberwas ten times larger than the number of corresponding target cells wereadded as PBMC groups, 100 μl per well. Blank wells and wells without theaddition of effector cells were set. AB11K was diluted to 50 μg/mL witha medium, after the 4-fold dilution, added to 96-well plates, 50 μl perwell, and incubated for 48 hours at 37° C. with 5% CO₂. The cell cultureplates were washed three times with PBS and the suspended cells wereremoved. A medium containing 10% CCK-8 was added, 100 μl per well, andincubated for 4 hours at 37° C. with 5% CO₂. Readings at 450 nm and 620nm were obtained. The specific killing rates of the antibodies werecalculated according to values at [OD450-OD620] using the formula asfollows:

${{Antibody}\mspace{14mu}{specific}\mspace{14mu}{killing}\mspace{14mu}{rate}\mspace{14mu}(\%)} = {\frac{\begin{matrix}\left\lbrack {{{OD}\; 450} - {{OD}\; 620}} \right\rbrack \\{\left( {{{effector}\mspace{14mu}{cells}} + {{target}\mspace{14mu}{cells}}} \right) - \left\lbrack {{{OD}\; 450} - {{OD}\; 620}} \right\rbrack} \\\left( {{antibody}\mspace{14mu}{treated}\mspace{14mu}{group}} \right)\end{matrix}}{\begin{matrix}\left\lbrack {{{OD}\; 450} - {{OD}\; 620}} \right\rbrack \\{\left( {{{effector}\mspace{14mu}{cells}} + {{target}\mspace{14mu}{cells}}} \right) - \left\lbrack {{{OD}\; 450} - {{OD}\; 620}} \right\rbrack} \\\left( {{blank}\mspace{14mu}{group}} \right)\end{matrix}} \times 100}$

The analysis was performed with the specific killing rate (%) as theY-axis and the antibody molar concentration as the X-axis throughsoftware GraphPad Prism 6 to calculate the EC₅₀ value for AB11K tomediate the killing on tumor target cells.

As shown in FIGS. 10-2 and 10-3 and Table 10-2, the bispecific antibodyAB11K exhibited very significant killing effects on tumor cells highlyexpressing Mucin1 through its mediation on effector cells. When expandedT cells were used as effector cells, the maximum specific killing ofAB11K reached 99% or more, wherein the specific killing effects onMCF-7, BT-549, HCC70 and T-47D were the best with EC₅₀ ranging from 100pM to 200 pM, followed by the specific killing effects on Hela, HCC1954and SK-OV-3, while the specific killing effect on HT-29 was the weakestwith a relatively large EC₅₀, about 1577 pM. When PBMCs were used aseffector cells, the specific killing effects of AB11K on MCF-7 andBT-549 were the best with the maximum specific killing of 95% or moreand EC₅₀ of 131.2 pM and 955.9 pM, respectively, followed by thespecific killing effects on HCC1954 and HCC70, and EC₅₀ for specificallykilling Hela and HT-29 were relatively large, which were 4810 pM and9550 pM, respectively.

TABLE 10-2 EC₅₀ results of AB11K to mediate effector cells to kill tumorcells T cell killing EC₅₀ PBMC killing EC₅₀ Cell name (pM) (pM) MCF-7152.7 131.2 BT-549 140.9 955.9 HCC70 185.4 595.2 T-47D 84.53 / HCC1954280.9 1893 Hela 278.2 4810 SK-OV-3 689.4 / HT-29 1577 9550

12.3 Evaluation of Abilities of Bispecific Antibodies to Activate TCells

Jurkat T cells containing NFAT RE reporter genes (purchased from BPSBioscience) can overexpress luciferase in the presence of bispecificantibodies and Mucin1-positive cells, and the degree of activation ofthe Jurkat T cells can be quantified by detecting the activity of theluciferase.

Specifically, cells MCF-7, BT-549, HCC70, T-47D, HCC1954, SK-OV-3, Helaand HT-29 were digested with trypsin, placed in 96-well cell cultureplates after the cell density of each kind of cells was adjusted to2×10⁵ cells/ml, 50 μl per well, and cultured overnight at 37° C. with 5%CO₂. The cell density of Jurkat-NFAT cells was adjusted to 2.5×10⁶cells/ml, 40 μl per well. AB11K was diluted to 400 μg/mL with a medium,after the 4-fold dilution, added to 96-well plates, 10 μl per well, andincubated for 48 hours at 37° C. with 5% CO₂ in an incubator.Steady-Glo® Luciferase was added, 100 μl per well and reacted for 5minutes. After that, the luminescence value was measured by a microplatereader. The analysis was performed with the fluorescein intensity as theY-axis and the antibody molar concentration as the X-axis throughsoftware GraphPad Prism 6 to calculate the EC₅₀ for bispecificantibodies to activate T cells.

As shown in FIG. 10-4 and Table 10-3, AB11K specifically activatedJurkat-NFAT cells, wherein the EC₅₀ value was at the nM level and itsconcentration was proportional to signal intensity.

TABLE 10-3 EC₅₀ results of the ability of AB11K to activate T cells Tcell activation EC₅₀ Cell name (nM) MCF-7 14.22 BT-549 10.49 HCC70 3.016T-47D 0.6294 HCC1954 5.599 Hela 7.241 SK-OV-3 10.37 HT-29 6.711

Example 13 Pharmacodynamics Study of Anti-EGFR×CD3 Bispecific Antibodiesin a Mouse Transplanted Tumor Model

A mouse transplanted tumor model of human skin cancer A431 cells thathighly expressed EGFR was selected to perform the pharmacodynamics studyof Anti-EGFR×CD3 bispecific antibodies AB8K, AB2K and Erbitux (fromMerck KGaA) on the in vivo inhibition of tumor growth.

CIK cells were prepared in the method as described in Example 3.1. A431cells in the logarithmic growth stage were collected. Female NPG mice atthe age of seven to eight weeks were selected, and 3×10⁶ A431 cells and1×10⁶ CIK cells were mixed and inoculated subcutaneously on the rightback of each NPG mouse. One hour later, the mice were randomly dividedinto five groups with six mice in each group according to their weightsand intraperitoneally administered with corresponding drugs. All treatedgroups and the PBS control group were administrated twice a week,wherein AB2K and Erbitux were administrated at a dose of 1 mg/kg. AB8Kwas administrated at doses of 1 mg/kg and 0.1 mg/kg. The day ofadministration was recorded as Day 0. The maximum diameter (D) and theminimum diameter (d) of the tumor were measured weekly. The volume (mm³)of the tumor of each group and the tumor growth inhibition rate (TGI)(%) of each treated group were calculated using the formulas as shown inExample 3.1.

As shown in FIG. 11, on Day 17 of administration, the average tumorvolume of the PBS control group was 1370.76±216.35 mm³; the averagetumor volume of the treated group administrated with Erbitux at a doseof 1 mg/kg was 1060.35±115.86 mm³, which was not significantly differentfrom that of the control group; the average tumor volume of the treatedgroup administrated with AB2K at a dose of 1 mg/kg was 877.76±120.38mm³, which was not significantly different from that of the controlgroup; the average tumor volumes of the treated groups administratedwith AB8K at doses of 0.1 mg/kg and 1 mg/kg were 233.30±135.51 mm³ and8.14±8.14 mm³, respectively, and TGIs were 82.98% and 98.36%,respectively, which were significantly different from that of thecontrol group (P<0.01), wherein the tumors in five of six mice of thetreated group administrated with AB8K at a dose of 1 mg/kg exhibitedcomplete regression. AB2K was an isotype control of AB8K. A431 cells didnot express CD20. AB2K exhibited no pharmacological effect in thismodel, indicating that the structure of the bispecific antibody isrelatively safe and does not cause non-specific killing. More than 90%of CIK cells were activated T cells. AB8K inhibited and killed tumorcells by activating human immune cells in animals, and completelyinhibited tumor growth at a dose of 1 mg/kg and exhibited a goodanti-tumor effect even at a dose of 0.1 mg/kg.

All the publications mentioned in the present invention are incorporatedherein by reference as if each publication is separately incorporatedherein by reference. In addition, it should be understood that thoseskilled in the art, who have read the disclosure, can make variouschanges or modifications on the present disclosure, and these equivalentforms fall within the scope of the appended claims.

1. A bispecific antibody, which is a tetravalent homodimer formed by twoidentical polypeptide chains that bind to each other by a covalent bond,wherein each of the polypeptide chains comprises a first single-chain Fvthat specifically binds to an tumor-associated antigen, a secondsingle-chain Fv that specifically bind to effector cell antigen CD3, andan Fc fragment in sequence from N-terminus to C-terminus; wherein thefirst single-chain Fv is linked to the second single-chain Fv by alinker peptide, the second single-chain Fv is linked to the Fc fragmentdirectly or by a linker peptide, and the Fc fragment has no effectorfunctions comprising CDC, ADCC, and ADCP.
 2. The bispecific antibodyaccording to claim 1, wherein the first single-chain Fv comprises a VHdomain and a VL domain that are linked by a linker peptide, which has anamino acid sequence of (GGGGX)_(n), wherein X comprises Ser or Ala, andn is a natural number of 1 to
 5. 3. The bispecific antibody according toclaim 1, wherein the tumor-associated antigen comprises CD19, CD20,CD22, CD25, CD30, CD33, CD38, CD39, CD40, CD47, CD52, CD73, CD74, CD123,CD133, CD138, BCMA, CA125, CEA, CS1, DLL3, DLL4, EGFR, EpCAM, FLT3,gpA33, GPC-3, Her2, MEGE-A3, NYESO1, PSMA, TAG-72, CIX, folate-bindingprotein, GD2, GD3, GM2, VEGF, VEGFR2, VEGFR3, Cadherin, Integrin,Mesothelin, Claudin18, αVβ3, α5β1, ERBB3, c-MET, IGF1R, EPHA3, TRAILR1,TRAILR2, RANKL, B7 protein family, Mucin family, FAP, and Tenascin. 4.The bispecific antibody according to claim 1, wherein the firstsingle-chain Fv specifically binds to CD19 and comprises a VH domain anda VL domain selected from the group consisting of: (i) a VH domaincomprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 9, 10, and11, respectively or having sequences that are substantially identical to(for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% ormore similar to or have one or more amino acid substitutions (forexample, conservative substitutions) than) any of SEQ ID NOs: 9, 10, and11; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQID NOs: 12, 13, and 14, respectively or having sequences that aresubstantially identical to (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) anyof SEQ ID NOs: 12, 13, and 14; (ii) a VH domain comprising HCDR1, HCDR2,and HCDR3 as shown in SEQ ID NOs: 17, 18, and 19, respectively or havingsequences that are substantially identical to (for example, are at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one ormore amino acid substitutions (for example, conservative substitutions)than) any of SEQ ID NOs: 17, 18, and 19; and a VL domain comprisingLCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 20, 21, and 22,respectively or having sequences that are substantially identical to(for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% ormore similar to or have one or more amino acid substitutions (forexample, conservative substitutions) than) any of SEQ ID NOs: 20, 21,and 22; (iii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown inSEQ ID NOs: 25, 26, and 27, respectively or having sequences that aresubstantially identical to (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) anyof SEQ ID NOs: 25, 26, and 27; and a VL domain comprising LCDR1, LCDR2,and LCDR3 as shown in SEQ ID NOs: 28, 29, and 30, respectively or havingsequences that are substantially identical to (for example, are at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one ormore amino acid substitutions (for example, conservative substitutions)than) any of SEQ ID NOs: 28, 29, and 30; and (iv) a VH domain comprisingHCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 33, 34, and 35,respectively or having sequences that are substantially identical to(for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% ormore similar to or have one or more amino acid substitutions (forexample, conservative substitutions) than) any of SEQ ID NOs: 33, 34,and 35; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown inSEQ ID NOs: 36, 37, and 38, respectively or having sequences that aresubstantially identical to (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) anyof SEQ ID NOs: 36, 37, and 38; or the first single-train Fv specificallybinds to CD20 and comprises a VH domain and a VL domain selected fromthe group consisting of: (i) a VH domain comprising HCDR1, HCDR2, andHCDR3 as shown in SEQ ID NOs: 41, 42, and 43, respectively or havingsequences that are substantially identical to (for example, are at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one ormore amino acid substitutions (for example, conservative substitutions)than) any of SEQ ID NOs: 41, 42, and 43; and a VL domain comprisingLCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 44, 45, and 46,respectively or having sequences that are substantially identical to(for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% ormore similar to or have one or more amino acid substitutions (forexample, conservative substitutions) than) any of SEQ ID NOs: 44, 45,and 46; (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown inSEQ ID NOs: 49, 50, and 51, respectively or having sequences that aresubstantially identical to (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) anyof SEQ ID NOs: 49, 50, and 51; and a VL domain comprising LCDR1, LCDR2,and LCDR3 as shown in SEQ ID NOs: 52, 53, and 54, respectively or havingsequences that are substantially identical to (for example, are at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one ormore amino acid substitutions (for example, conservative substitutions)than) any of SEQ ID NOs: 52, 53, and 54; (iii) a VH domain comprisingHCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 57, 58, and 59,respectively or having sequences that are substantially identical to(for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% ormore similar to or have one or more amino acid substitutions (forexample, conservative substitutions) than) any of SEQ ID NOs: 57, 58,and 59; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown inSEQ ID NOs: 60, 61, and 62, respectively or having sequences that aresubstantially identical to (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) anyof SEQ ID NOs: 60, 61, and 62; and (iv) a VH domain comprising HCDR1,HCDR2, and HCDR3 as shown in SEQ ID NOs: 65, 66, and 67, respectively orhaving sequences that are substantially identical to (for example, areat least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to orhave one or more amino acid substitutions (for example, conservativesubstitutions) than) any of SEQ ID NOs: 33, 34, and 35; and a VL domaincomprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 68, 69, and70, respectively or having sequences that are substantially identical to(for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% ormore similar to or have one or more amino acid substitutions (forexample, conservative substitutions) than) any of SEQ ID NOs: 68, 69,and 70; or the first single-train Fv specifically binds to CD22 andcomprises a VH domain and a VL domain selected from the group consistingof: (i) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQID NOs: 73, 74, and 75, respectively or having sequences that aresubstantially identical to (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) anyof SEQ ID NOs: 73, 74, and 75; and a VL domain comprising LCDR1, LCDR2,and LCDR3 as shown in SEQ ID NOs: 76, 77, and 78, respectively or havingsequences that are substantially identical to (for example, are at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one ormore amino acid substitutions (for example, conservative substitutions)than) any of SEQ ID NOs: 76, 77, and 78; (ii) a VH domain comprisingHCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 81, 82, and 83,respectively or having sequences that are substantially identical to(for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% ormore similar to or have one or more amino acid substitutions (forexample, conservative substitutions) than) any of SEQ ID NOs: 81, 82,and 83; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown inSEQ ID NOs: 84, 85, and 86, respectively or having sequences that aresubstantially identical to (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) anyof SEQ ID NOs: 84, 85, and 86; or the first single-train Fv specificallybinds to CD30 and comprises a VH domain and a VL domain selected fromthe group consisting of: (i) a VH domain comprising HCDR1, HCDR2, andHCDR3 as shown in SEQ ID NOs: 89, 90, and 91, respectively or havingsequences that are substantially identical to (for example, are at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one ormore amino acid substitutions (for example, conservative substitutions)than) any of SEQ ID NOs: 89, 90, and 91; and a VL domain comprisingLCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 92, 93, and 94,respectively or having sequences that are substantially identical to(for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% ormore similar to or have one or more amino acid substitutions (forexample, conservative substitutions) than) any of SEQ ID NOs: 92, 93,and 94; and (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shownin SEQ ID NOs: 97, 98, and 99, respectively or having sequences that aresubstantially identical to (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) anyof SEQ ID NOs: 97, 98, and 99; and a VL domain comprising LCDR1, LCDR2,and LCDR3 as shown in SEQ ID NOs: 100, 101, and 102, respectively orhaving sequences that are substantially identical to (for example, areat least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to orhave one or more amino acid substitutions (for example, conservativesubstitutions) than) any of SEQ ID NOs: 100, 101, and 102; or the firstsingle-train Fv specifically binds to ECAM and comprises a VH domain anda VL domain selected from the group consisting of: (i) a VH domaincomprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 105, 106, and107, respectively or having sequences that are substantially identicalto (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% ormore similar to or have one or more amino acid substitutions (forexample, conservative substitutions) than) any of SEQ ID NOs: 105, 106,and 107; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown inSEQ ID NOs: 108, 109, and 110, respectively or having sequences that aresubstantially identical to (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) anyof SEQ ID NOs: 108, 109, and 110; and (ii) a VH domain comprising HCDR1,HCDR2, and HCDR3 as shown in SEQ ID NOs: 113, 114, and 115, respectivelyor having sequences that are substantially identical to (for example,are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar toor have one or more amino acid substitutions (for example, conservativesubstitutions) than) any of SEQ ID NOs: 113, 114, and 115; and a VLdomain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 116,117, and 118, respectively or having sequences that are substantiallyidentical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or more similar to or have one or more amino acid substitutions(for example, conservative substitutions) than) any of SEQ ID NOs: 116,117, and 118; or the first single-train Fv specifically binds to CEA andcomprises a VH domain and a VL domain selected from the group consistingof: (i) a VH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQID NOs: 121, 122, and 123, respectively or having sequences that aresubstantially identical to (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) anyof SEQ ID NOs: 121, 122, and 123; and a VL domain comprising LCDR1,LCDR2, and LCDR3 as shown in SEQ ID NOs: 124, 125, and 126, respectivelyor having sequences that are substantially identical to (for example,are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar toor have one or more amino acid substitutions (for example, conservativesubstitutions) than) any of SEQ ID NOs: 124, 125, and 126; (ii) a VHdomain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 129,130, and 131, respectively or having sequences that are substantiallyidentical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or more similar to or have one or more amino acid substitutions(for example, conservative substitutions) than) any of SEQ ID NOs: 129,130, and 131; and a VL domain comprising LCDR1, LCDR2, and LCDR3 asshown in SEQ ID NOs: 132, 133, and 134, respectively or having sequencesthat are substantially identical to (for example, are at least 80%, 85%,90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or moreamino acid substitutions (for example, conservative substitutions) than)any of SEQ ID NOs: 132, 133, and 134; (iii) a VH domain comprisingHCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 137, 138, and 139,respectively or having sequences that are substantially identical to(for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% ormore similar to or have one or more amino acid substitutions (forexample, conservative substitutions) than) any of SEQ ID NOs: 137, 138,and 139; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown inSEQ ID NOs: 140, 141, and 142, respectively or having sequences that aresubstantially identical to (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) anyof SEQ ID NOs: 140, 141, and 142; or the first single-train Fvspecifically binds to Her2 and comprises a VH domain and a VL domainselected from the group consisting of: (i) a VH domain comprising HCDR1,HCDR2, and HCDR3 as shown in SEQ ID NOs: 145, 146, and 147, respectivelyor having sequences that are substantially identical to (for example,are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar toor have one or more amino acid substitutions (for example, conservativesubstitutions) than) any of SEQ ID NOs: 145, 146, and 147; and a VLdomain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 148,149, and 150, respectively or having sequences that are substantiallyidentical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or more similar to or have one or more amino acid substitutions(for example, conservative substitutions) than) any of SEQ ID NOs: 148,149, and 150; (ii) a VH domain comprising HCDR1, HCDR2, and HCDR3 asshown in SEQ ID NOs: 153, 154, and 155, respectively or having sequencesthat are substantially identical to (for example, are at least 80%, 85%,90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or moreamino acid substitutions (for example, conservative substitutions) than)any of SEQ ID NOs: 153, 154, and 155; and a VL domain comprising LCDR1,LCDR2, and LCDR3 as shown in SEQ ID NOs: 156, 157, and 158, respectivelyor having sequences that are substantially identical to (for example,are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar toor have one or more amino acid substitutions (for example, conservativesubstitutions) than) any of SEQ ID NOs: 156, 157, and 158; and (iii) aVH domain comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs:161, 162, and 163, respectively or having sequences that aresubstantially identical to (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) anyof SEQ ID NOs: 16, 162, and 163; and a VL domain comprising LCDR1,LCDR2, and LCDR3 as shown in SEQ ID NOs: 164, 165, and 166, respectivelyor having sequences that are substantially identical to (for example,are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar toor have one or more amino acid substitutions (for example, conservativesubstitutions) than) any of SEQ ID NOs: 164, 165, and 166; or the firstsingle-train Fv specifically binds to EGFR and comprises a VH domain anda VL domain selected from the group consisting of: (i) a VH domaincomprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 169, 170, and171, respectively or having sequences that are substantially identicalto (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% ormore similar to or have one or more amino acid substitutions (forexample, conservative substitutions) than) any of SEQ ID NOs: 169, 170,and 171; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown inSEQ ID NOs: 172, 173, and 174, respectively or having sequences that aresubstantially identical to (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) anyof SEQ ID NOs: 172, 173, and 174; (ii) a VH domain comprising HCDR1,HCDR2, and HCDR3 as shown in SEQ ID NOs: 177, 178, and 179, respectivelyor having sequences that are substantially identical to (for example,are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar toor have one or more amino acid substitutions (for example, conservativesubstitutions) than) any of SEQ ID NOs: 177, 178, and 179; and a VLdomain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 180,181, and 182, respectively or having sequences that are substantiallyidentical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or more similar to or have one or more amino acid substitutions(for example, conservative substitutions) than) any of SEQ ID NOs: 180,181, and 182; and (iii) a VH domain comprising HCDR1, HCDR2, and HCDR3as shown in SEQ ID NOs: 185, 186, and 187, respectively or havingsequences that are substantially identical to (for example, are at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one ormore amino acid substitutions (for example, conservative substitutions)than) any of SEQ ID NOs: 185, 186, and 187; and a VL domain comprisingLCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 188, 189, and 190,respectively or having sequences that are substantially identical to(for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% ormore similar to or have one or more amino acid substitutions (forexample, conservative substitutions) than) any of SEQ ID NOs: 188, 189,and 190; or the first single-train Fv specifically binds to GPC-3 andcomprises a VH domain and a VL domain selected from: a VH domaincomprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 193, 194, and195, respectively or having sequences that are substantially identicalto (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% ormore similar to or have one or more amino acid substitutions (forexample, conservative substitutions) than) any of SEQ ID NOs: 193, 194,and 195; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown inSEQ ID NOs: 196, 197, and 198, respectively or having sequences that aresubstantially identical to (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) anyof SEQ ID NOs: 196, 197, and 198; or the first single-train Fvspecifically binds to Mesothelin and comprises a VH domain and a VLdomain selected from: a VH domain comprising HCDR1, HCDR2, and HCDR3 asshown in SEQ ID NOs: 201, 202, and 203, respectively or having sequencesthat are substantially identical to (for example, are at least 80%, 85%,90%, 92%, 95%, 97%, 98%, 99% or more similar to or have one or moreamino acid substitutions (for example, conservative substitutions) than)any of SEQ ID NOs: 201, 202, and 203; and a VL domain comprising LCDR1,LCDR2, and LCDR3 as shown in SEQ ID NOs: 204, 205, and 206, respectivelyor having sequences that are substantially identical to (for example,are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar toor have one or more amino acid substitutions (for example, conservativesubstitutions) than) any of SEQ ID NOs: 204, 205, and 206; or the firstsingle-train Fv specifically binds to Mucin1 and comprises a VH domainand a VL domain selected from the group consisting of: (i) a VH domaincomprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 209, 210, and211, respectively or having sequences that are substantially identicalto (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% ormore similar to or have one or more amino acid substitutions (forexample, conservative substitutions) than) any of SEQ ID NOs: 209, 210,and 211; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown inSEQ ID NOs: 212, 213, and 214, respectively or having sequences that aresubstantially identical to (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) anyof SEQ ID NOs: 212, 213, and 214; and (ii) a VH domain comprising HCDR1,HCDR2, and HCDR3 as shown in SEQ ID NOs: 217, 218, and 219, respectivelyor having sequences that are substantially identical to (for example,are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar toor have one or more amino acid substitutions (for example, conservativesubstitutions) than) any of SEQ ID NOs: 217, 218, and 219; and a VLdomain comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 220,221, and 222, respectively or having sequences that are substantiallyidentical to (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or more similar to or have one or more amino acid substitutions(for example, conservative substitutions) than) any of SEQ ID NOs: 220,221, and 2222; or the first single-train Fv specifically binds to CA125and comprises a VH domain and a VL domain selected from: a VH domaincomprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 225, 226, and227, respectively or having sequences that are substantially identicalto (for example, are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% ormore similar to or have one or more amino acid substitutions (forexample, conservative substitutions) than) any of SEQ ID NOs: 225, 226,and 227; and a VL domain comprising LCDR1, LCDR2, and LCDR3 as shown inSEQ ID NOs: 228, 229, and 230, respectively or having sequences that aresubstantially identical the (for example, are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions (for example, conservative substitutions) than) anyof SEQ ID NOs: 228, 229, and
 230. 5-15. (canceled)
 16. The bispecificantibody according to claim 1, wherein the first single-chain Fvspecifically binds to CD19 and comprises a VH domain and a VL domainselected from the group consisting of: (i) a VH domain comprising anamino acid sequence as shown in SEQ ID NO: 15 or having a sequence thatis substantially identical to (for example, is at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:15; and a VL domain comprising an amino acid sequence as shown in SEQ IDNO: 16 or having a sequence that is substantially identical to (forexample, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or moresimilar to or has one or more amino acid substitutions (for example,conservative substitutions) than) SEQ ID NO: 16; (ii) a VH domaincomprising an amino acid sequence as shown in SEQ ID NO: 23 or having asequence that is substantially identical to (for example, is at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one ormore amino acid substitutions (for example, conservative substitutions)than) SEQ ID NO: 23; and a VL domain comprising an amino acid sequenceas shown in SEQ ID NO: 24 or having a sequence that is substantiallyidentical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or more similar to or has one or more amino acid substitutions(for example, conservative substitutions) than) SEQ ID NO: 24; (iii) aVH domain comprising an amino acid sequence as shown in SEQ ID NO: 31 orhaving a sequence that is substantially identical (for example, is atleast 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or hasone or more amino acid substitutions (for example, conservativesubstitutions) than) to SEQ ID NO: 31; and a VL domain comprising anamino acid sequence as shown in SEQ ID NO: 32 or having a sequence thatis substantially identical to (for example, is at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:32; and (iv) a VH domain comprising an amino acid sequence as shown inSEQ ID NO: 39 or having a sequence that is substantially identical to(for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or moresimilar to or has one or more amino acid substitutions (for example,conservative substitutions) than) SEQ ID NO: 39; and a VL domaincomprising an amino acid sequence as shown in SEQ ID NO: 40 or having asequence that is substantially identical to (for example, is at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one ormore amino acid substitutions (for example, conservative substitutions)than) SEQ ID NO: 40; or the first single-chain Fv specifically binds toCD20 and comprises a VH domain and a VL domain selected from the groupconsisting of: (i) a VH domain comprising an amino acid sequence asshown in SEQ ID NO: 47 or having a sequence that is substantiallyidentical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or more similar to or has one or more amino acid substitutions(for example, conservative substitutions) than) SEQ ID NO: 15; and a VLdomain comprising an amino acid sequence as shown in SEQ ID NO: 48 orhaving a sequence that is substantially identical to (for example, is atleast 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or hasone or more amino acid substitutions (for example, conservativesubstitutions) than) SEQ ID NO: 48; (ii) a VH domain comprising an aminoacid sequence as shown in SEQ ID NO: 55 or having a sequence that issubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:55; and a VL domain comprising an amino acid sequence as shown in SEQ IDNO: 24 or having a sequence that is substantially identical to (forexample, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or moresimilar to or has one or more amino acid substitutions (for example,conservative substitutions) than) SEQ ID NO: 56; (iii) a VH domaincomprising an amino acid sequence as shown in SEQ ID NO: 63 or having asequence that is substantially identical (for example, is at least 80%,85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or moreamino acid substitutions (for example, conservative substitutions) than)to SEQ ID NO: 63; and a VL domain comprising an amino acid sequence asshown in SEQ ID NO: 64 or having a sequence that is substantiallyidentical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or more similar to or has one or more amino acid substitutions(for example, conservative substitutions) than) SEQ ID NO: 64; and (iv)a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 71or having a sequence that is substantially identical to (for example, isat least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to orhas one or more amino acid substitutions (for example, conservativesubstitutions) than) SEQ ID NO: 71; and a VL domain comprising an aminoacid sequence as shown in SEQ ID NO: 72 or having a sequencesubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:72; or the first single-chain Fv specifically binds to CD22 andcomprises a VH domain and a VL domain selected from the group consistingof: (i) a VH domain comprising an amino acid sequence as shown in SEQ IDNO: 79 or having a sequence that is substantially identical to (forexample, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or moresimilar to or has one or more amino acid substitutions (for example,conservative substitutions) than) SEQ ID NO: 79; and a VL domaincomprising an amino acid sequence as shown in SEQ ID NO: 80 or having asequence that is substantially identical to (for example, is at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one ormore amino acid substitutions (for example, conservative substitutions)than) SEQ ID NO: 80; (ii) a VH domain comprising an amino acid sequenceas shown in SEQ ID NO: 87 or having a sequence that is substantiallyidentical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or more similar to or has one or more amino acid substitutions(for example, conservative substitutions) than) SEQ ID NO: 87; and a VLdomain comprising an amino acid sequence as shown in SEQ ID NO: 88 orhaving a sequence that is substantially identical to (for example, is atleast 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or hasone or more amino acid substitutions (for example, conservativesubstitutions) than) SEQ ID NO: 88; or the first single-chain Fvspecifically binds to CD30 and comprises a VH domain and a VL domainselected from the group consisting of: (i) a VH domain comprising anamino acid sequence as shown in SEQ ID NO: 95 or having a sequence thatis substantially identical to (for example, is at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:95; and a VL domain comprising an amino acid sequence as shown in SEQ IDNO: 96 or having a sequence that is substantially identical to (forexample, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or moresimilar to or has one or more amino acid substitutions (for example,conservative substitutions) than) SEQ ID NO: 96; and (ii) a VH domaincomprising an amino acid sequence as shown in SEQ ID NO: 103 or having asequence that is substantially identical to (for example, is at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one ormore amino acid substitutions (for example, conservative substitutions)than) SEQ ID NO: 103; and a VL domain comprising an amino acid sequenceas shown in SEQ ID NO: 104 or having a sequence that is substantiallyidentical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or more similar to or has one or more amino acid substitutions(for example, conservative substitutions) than) SEQ ID NO: 104; or thefirst single-chain Fv specifically binds to EpCAM and comprises a VHdomain and a VL domain selected from the group consisting of: (i) a VHdomain comprising an amino acid sequence as shown in SEQ ID NO: 111 orhaving a sequence that is substantially identical to (for example, is atleast 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or hasone or more amino acid substitutions (for example, conservativesubstitutions) than) SEQ ID NO: 111; and a VL domain comprising an aminoacid sequence as shown in SEQ ID NO: 112 or having a sequence that issubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:112; and (ii) a VH domain comprising an amino acid sequence as shown inSEQ ID NO: 119 or having a sequence that is substantially identical to(for example, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or moresimilar to or has one or more amino acid substitutions (for example,conservative substitutions) than) SEQ ID NO: 119; and a VL domaincomprising an amino acid sequence as shown in SEQ ID NO: 120 or having asequence that is substantially identical to (for example, is at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one ormore amino acid substitutions (for example, conservative substitutions)than) SEQ ID NO: 120; or the first single-chain Fv specifically binds toCEA and comprises a VH domain and a VL domain selected from the groupconsisting of: (i) a VH domain comprising an amino acid sequence asshown in SEQ ID NO: 127 or having a sequence that is substantiallyidentical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or more similar to or has one or more amino acid substitutions(for example, conservative substitutions) than) SEQ ID NO: 127; and a VLdomain comprising an amino acid sequence as shown in SEQ ID NO: 128 orhaving a sequence that is substantially identical to (for example, is atleast 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or hasone or more amino acid substitutions (for example, conservativesubstitutions) than) SEQ ID NO: 128; (ii) a VH domain comprising anamino acid sequence as shown in SEQ ID NO: 135 or having a sequence thatis substantially identical to (for example, is at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:135; and a VL domain comprising an amino acid sequence as shown in SEQID NO: 136 or having a sequence that is substantially identical to (forexample, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or moresimilar to or has one or more amino acid substitutions (for example,conservative substitutions) than) SEQ ID NO: 136; and (iii) a VH domaincomprising an amino acid sequence as shown in SEQ ID NO: 143 or having asequence that is substantially identical (for example, is at least 80%,85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one or moreamino acid substitutions (for example, conservative substitutions) than)SEQ ID NO: 143; and a VL domain comprising an amino acid sequence asshown in SEQ ID NO: 144 or having a sequence that is substantiallyidentical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or more similar to or has one or more amino acid substitutions(for example, conservative substitutions) than) SEQ ID NO: 144; or thefirst single-chain Fv specifically binds to Her2 and comprises a VHdomain and a VL domain selected from the group consisting of: (i) a VHdomain comprising an amino acid sequence as shown in SEQ ID NO: 151 orhaving a sequence that is substantially identical to (for example, is atleast 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or hasone or more amino acid substitutions (for example, conservativesubstitutions) than) SEQ ID NO: 151; and a VL domain comprising an aminoacid sequence as shown in SEQ ID NO: 152 or having a sequence that issubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:152; (ii) a VH domain comprising an amino acid sequence as shown in SEQID NO: 159 or having a sequence that is substantially identical to (forexample, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or moresimilar to or has one or more amino acid substitutions (for example,conservative substitutions) than) SEQ ID NO: 159; and a VL domaincomprising an amino acid sequence as shown in SEQ ID NO: 160 or having asequence that is substantially identical to (for example, is at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one ormore amino acid substitutions (for example, conservative substitutions)than) SEQ ID NO: 160; and (iii) a VH domain comprising an amino acidsequence as shown in SEQ ID NO: 167 or having a sequence that issubstantially identical (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:167; and a VL domain comprising an amino acid sequence as shown in SEQID NO: 168 or having a sequence that is substantially identical to (forexample, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or moresimilar to or has one or more amino acid substitutions (for example,conservative substitutions) than) SEQ ID NO: 168; or the firstsingle-chain Fv specifically binds to EGFR and comprises a VH domain anda VL domain selected from the group consisting of: (i) a VH domaincomprising an amino acid sequence as shown in SEQ ID NO: 175 or having asequence that is substantially identical to (for example, is at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one ormore amino acid substitutions (for example, conservative substitutions)than) SEQ ID NO: 175; and a VL domain comprising an amino acid sequenceas shown in SEQ ID NO: 152 or having a sequence that is substantiallyidentical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or more similar to or has one or more amino acid substitutions(for example, conservative substitutions) than) SEQ ID NO: 176; (ii) aVH domain comprising an amino acid sequence as shown in SEQ ID NO: 183or having a sequence that is substantially identical to (for example, isat least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to orhas one or more amino acid substitutions (for example, conservativesubstitutions) than) SEQ ID NO: 183; and a VL domain comprising an aminoacid sequence as shown in SEQ ID NO: 184 or having a sequence that issubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:184; and (iii) a VH domain comprising an amino acid sequence as shown inSEQ ID NO: 191 or having a sequence that is substantially identical (forexample, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or moresimilar to or has one or more amino acid substitutions (for example,conservative substitutions) than) SEQ ID NO: 191; and a VL domaincomprising an amino acid sequence as shown in SEQ ID NO: 168 or having asequence that is substantially identical to (for example, is at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one ormore amino acid substitutions (for example, conservative substitutions)than) SEQ ID NO: 192; or the first single-chain Fv specifically binds toGPC-3 and comprises a VH domain and a VL domain selected from: a VHdomain comprising an amino acid sequence as shown in SEQ ID NO: 199 orhaving a sequence substantially identical to (for example, is at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one ormore amino acid substitutions (for example, conservative substitutions)than) SEQ ID NO: 199; and a VL domain comprising an amino acid sequenceas shown in SEQ ID NO: 200 or having a sequence that is substantiallyidentical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or more similar to or has one or more amino acid substitutions(for example, conservative substitutions) than) SEQ ID NO: 200; or thefirst single-chain Fv specifically binds to Mesothelin and comprises aVH domain and a VL domain selected from: a VH domain comprising an aminoacid sequence as shown in SEQ ID NO: 207 or having a sequence that issubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:207; and a VL domain comprising an amino acid sequence as shown in SEQID NO: 208 or having a sequence that is substantially identical to (forexample, is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or moresimilar to or has one or more amino acid substitutions (for example,conservative substitutions) than) SEQ ID NO: 208; or the firstsingle-chain Fv specifically binds to Mucin1 and comprises a VH domainand a VL domain selected from the group consisting of: (i) a VH domaincomprising an amino acid sequence as shown in SEQ ID NO: 215 or having asequence that is substantially identical to (for example, is at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one ormore amino acid substitutions (for example, conservative substitutions)than) SEQ ID NO: 215; and a VL domain comprising an amino acid sequenceas shown in SEQ ID NO: 216 or having a sequence that is substantiallyidentical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or more similar to or has one or more amino acid substitutions(for example, conservative substitutions) than) SEQ ID NO: 216; and (ii)a VH domain comprising an amino acid sequence as shown in SEQ ID NO: 223or having a sequence that is substantially identical to (for example, isat least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to orhas one or more amino acid substitutions (for example, conservativesubstitutions) than) SEQ ID NO: 159; and a VL domain comprising an aminoacid sequence as shown in SEQ ID NO: 160 or having a sequence that issubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:224; and or the first single-chain Fv specifically binds to CA125 andcomprises a VH domain and a VL domain selected from: a VH domaincomprising an amino acid sequence as shown in SEQ ID NO: 231 or having asequence that is substantially identical to (for example, is at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one ormore amino acid substitutions (for example, conservative substitutions)than) SEQ ID NO: 231; and a VL domain comprising an amino acid sequenceas shown in SEQ ID NO: 232 or having a sequence that is substantiallyidentical to (for example, is at least 80%, 85%, 90%, 92%, 95%, 97%,98%, 99% or more similar to or has one or more amino acid substitutions(for example, conservative substitutions) than) SEQ ID NO:
 232. 17-27.(canceled)
 28. The bispecific antibody according to claim 1, wherein thesecond single-chain Fv comprises a VH domain and a VL domain that arelinked by a linker peptide which has an amino acid sequence of(GGGGX)_(n), wherein X comprises Ser or Ala, preferably Ser, and n is anatural number of 1 to 5, preferably 3, wherein the single chain Fvbinds to an effector cell at an EC₅₀ value greater than about 50 nM, orgreater than 100 nM, or greater than 300 nM, or greater than 500 nM inan in vitro binding affinity assay; and wherein, the second single-chainFv of the bispecific antibody is capable of binding to human CD3 andspecifically binding to CD3 of a cynomolgus monkey or a rhesus monkey.29. (canceled)
 30. The bispecific antibody according to claim 28,wherein the second single-chain Fv comprises a VH domain comprisingHCDR1, HCDR2, and HCDR3 as shown in SEQ ID NOs: 241, 242, and 243,respectively or having sequences that are at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or have one or more amino acidsubstitutions than SEQ ID NOs: 241, 242, and 243; and a VL domaincomprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NOs: 244, 245, and246, respectively or having sequences that are at least 80%, 85%, 90%,92%, 95%, 97%, 98%, 99% or more similar to or have one or more aminoacid substitutions than SEQ ID NOs: 244, 245, and 246, or the secondsingle-chain Fv comprises a VH domain comprising HCDR1, HCDR2, and HCDR3as shown in SEQ ID NOs: 249, 250, and 251, respectively or havingsequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% ormore similar to or have one or more amino acid substitutions than SEQ IDNOs: 249, 250, and 251; and a VL domain comprising LCDR1, LCDR2, andLCDR3 as shown in SEQ ID NOs: 252, 253, and 254, respectively or havingsequences that are at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% ormore similar to or have one or more amino acid substitutions than any ofSEQ ID NOs: 252, 253, and
 254. 31. (canceled)
 32. The bispecificantibody according to claim 30, wherein the second single-chain Fvspecifically binds to CD3; the VH domain of the second single-chain Fvcomprises an amino acid sequence as shown in SEQ ID NO: 247 or having asequence that is substantially identical to (for example, is at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one ormore amino acid substitutions (for example, conservative substitutions)than) SEQ ID NO: 247; and the VL domain of the second single-chain Fvcomprises an amino acid sequence as shown in SEQ ID NO: 248 or having asequence that is substantially identical to (for example, is at least80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more similar to or has one ormore amino acid substitutions (for example, conservative substitutions)than) SEQ ID NO: 248; or the second single-chain Fv specifically bindsto CD3; the VH domain of the second single-chain Fv comprises an aminoacid sequence as show in SEQ ID NO: 255 or having a sequence that issubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99%, or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than) SEQ ID NO:255; and the VL domain of the second single-chain Fv comprises an aminoacid sequence as shown in SEQ ID NO: 256 or having a sequence that issubstantially identical to (for example, is at least 80%, 85%, 90%, 92%,95%, 97%, 98%, 99% or more similar to or has one or more amino acidsubstitutions (for example, conservative substitutions) than SEQ ID NO:256.
 33. (canceled)
 34. The bispecific antibody according to claim 31,wherein the linker peptide that links the first single-chain Fv to thesecond single-chain Fv consists of a flexible peptide and a rigidpeptide; wherein the flexible peptide comprises two or more amino acids,and preferably selected from the following amino acids: Gly(G), Ser(S),Ala(A), and Thr(T); more preferably, the flexible peptide comprises Gand S residues; most preferably, an amino acid composition structure ofthe flexible peptide has a general formula of G_(x)S_(y)(GGGGS)_(z),wherein x, y, and z are integers greater than or equal to 0, andx+y+z≥1; the rigid peptide is derived from a full-length sequenceconsisting of amino acids at positions 118 to 145 at carboxyl terminusof the natural human chorionic gonadotropin beta-subunit, or a truncatedfragment thereof; preferably, the rigid peptide comprises SSSSKAPPPS.35. The bispecific antibody according to claim 34, wherein the linkerpeptide comprises an amino acid sequence as shown in SEQ ID NO:
 258. 36.The bispecific antibody according to claim 1, wherein the linker peptidethat links the Fc fragment to the second single-chain Fv comprises 1 to20 amino acids, and preferably selected from the following amino acids:Gly(G), Ser(S), Ala(A), and Thr(T); more preferably Gly(G) and Ser(S);further preferably, the linker peptide consists of (GGGGS)_(n), whereinn=1, 2, 3 or
 4. 37. The bispecific antibody according to claim 1,wherein the Fc fragment comprises a hinge region, a CH2 domain, and aCH3 domain from a human immunoglobulin heavy chain constant region;preferably, the Fc fragment is selected from heavy chain constantregions of human IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE;more preferably, the Fc fragment is selected from heavy chain constantregions of human IgG1, IgG2, IgG3, and IgG4; further preferably, the Fcfragment is selected from heavy chain constant region of human IgG1 orIgG4; and compared to a natural sequence from which the Fc fragment isderived, the Fc fragment has one or more amino acid substitutions,deletions or additions selected from the group consisting of: (i) aminoacid substitutions L234A/L235A/P331S that are determined according to anEU numbering system (ii) amino acid substitutions M428L, T250Q/M428L,M248L/N434S or M252Y/S254T/T25E determined according to the EU numberingsystem; (iii) amino acid substitution N297A determined according to theEU numbering system; and (iv) an amino acid deletion K447 determinedaccording to the EU numbering system. 38-41. (canceled)
 42. Thebispecific antibody according to claim 37, wherein the Fc fragment hasan amino acid sequence as shown in SEQ ID NO: 263 that has six aminoacid substitutions or replacements L234A/L235A/N297A/P331S/T250Q/M428Ldetermined according to the EU numbering system and a deleted or removedK447 determined according to the EU numbering system compared to thenatural sequence from which the Fc fragment is derived.
 43. Thebispecific antibody according to claim 1, wherein the bispecificantibody binds to human CD19 and CD3 and has an amino acid sequence asfollows: (i) a sequence as shown in SEQ ID NO: 264; (ii) a sequence withone or more substitutions, deletions or additions (such as 1, 2, 3, 4 or5 substitutions, deletions or additions) compared to the sequence asshown in SEQ ID NO: 264; or (iii) a sequence with at least 80%, at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% or 100% sequence identity to the sequence as shown in SEQ ID NO:264; or the bispecific antibody binds to human CD19 and CD3 and has anamino acid sequence as follows: (i) a sequence as shown in SEQ ID NO:283; (ii) a sequence with one or more substitutions, deletions oradditions (such as 1, 2, 3, 4 or 5 substitutions, deletions oradditions) compared to the sequence as shown in SEQ ID NO: 283; or (iii)a sequence with at least 80%, at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% sequence identity to thesequence as shown in SEQ ID NO: 283; or the bispecific antibody binds tohuman CD20 and CD3 and has an amino acid sequence as follows: (i) asequence as shown in SEQ ID NO: 266; (ii) a sequence with one or moresubstitutions, deletions or additions (such as 1, 2, 3, 4 or 5substitutions, deletions or additions) compared to the sequence as shownin SEQ ID NO: 266; or (iii) a sequence with at least 80%, at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to the sequence as shown in SEQ ID NO: 266; orthe bispecific antibody binds to human CD22 and CD3 and has an aminoacid sequence as follows: (i) a sequence as shown in SEQ ID NO: 268;(ii) a sequence with one or more substitutions, deletions or additions(such as 1, 2, 3, 4 or 5 substitutions, deletions or additions) comparedto the sequence as shown in SEQ ID NO: 268; or (iii) a sequence with atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to the sequence asshown in SEQ ID NO: 268; or the bispecific antibody binds to human CD30and CD3 and has an amino acid sequence as follows: (i) a sequence asshown in SEQ ID NO: 270; (ii) a sequence with one or more substitutions,deletions or additions (such as 1, 2, 3, 4 or 5 substitutions, deletionsor additions) compared to the sequence as shown in SEQ ID NO: 270; or(iii) a sequence with at least 80%, at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the sequence as shown in SEQ ID NO: 270; or the bispecific antibodybinds to human EpCAM and CD3 and has an amino acid sequence as follows:(i) a sequence as shown in SEQ ID NO: 272; (ii) a sequence with one ormore substitutions, deletions or additions (such as 1, 2, 3, 4 or 5substitutions, deletions or additions) compared to the sequence as shownin SEQ ID NO: 272; or (iii) a sequence with at least 80%, at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to the sequence as shown in SEQ ID NO: 272; orthe bispecific antibody binds to human CEA and CD3 and has an amino acidsequence as follows: (i) a sequence as shown in SEQ ID NO: 274; (ii) asequence with one or more substitutions, deletions or additions (such as1, 2, 3, 4 or 5 substitutions, deletions or additions) compared to thesequence as shown in SEQ ID NO: 274; or (iii) a sequence with at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to the sequence as shown inSEQ ID NO: 274; or the bispecific antibody binds to human Her2 and CD3and has an amino acid sequence as follows: (i) a sequence as shown inSEQ ID NO: 8; (ii) a sequence with one or more substitutions, deletionsor additions (such as 1, 2, 3, 4 or 5 substitutions, deletions oradditions) compared to the sequence as shown in SEQ ID NO: 8; or (iii) asequence with at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% sequence identity to thesequence as shown in SEQ ID NO: 8; or the bispecific antibody binds tohuman EGFR and CD3 and has an amino acid sequence as follows: (i) asequence as shown in SEQ ID NO: 277; (ii) a sequence with one or moresubstitutions, deletions or additions (such as 1, 2, 3, 4 or 5substitutions, deletions or additions) compared to the sequence as shownin SEQ ID NO: 277; or (iii) a sequence with at least 80%, at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to the sequence as shown in SEQ ID NO: 277; orthe bispecific antibody binds to human GPC-3 and CD3 and has an aminoacid sequence as follows: (i) a sequence as shown in SEQ ID NO: 279;(ii) a sequence with one or more substitutions, deletions or additions(such as 1, 2, 3, 4 or 5 substitutions, deletions or additions) comparedto the sequence as shown in SEQ ID NO: 279; or (iii) a sequence with atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to the sequence asshown in SEQ ID NO: 279; or the bispecific antibody binds to humanMesothelin and CD3 and has an amino acid sequence as follows: (i) asequence as shown in SEQ ID NO: 281; (ii) a sequence with one or moresubstitutions, deletions or additions (such as 1, 2, 3, 4 or 5substitutions, deletions or additions) compared to the sequence as shownin SEQ ID NO: 281; or (iii) a sequence with at least 80%, at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% sequence identity to the sequence as shown in SEQ ID NO: 281; orthe bispecific antibody binds to human Mucin1 and CD3 and has an aminoacid sequence as follows: (i) a sequence as shown in SEQ ID NO: 285;(ii) a sequence with one or more substitutions, deletions or additions(such as 1, 2, 3, 4 or 5 substitutions, deletions or additions) comparedto the sequence as shown in SEQ ID NO: 285; or (iii) a sequence with atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% sequence identity to the sequence asshown in SEQ ID NO:
 285. 44-54. (canceled)
 55. A DNA molecule encodingthe bispecific antibody according to claim 1, which has a nucleotidesequence as shown in SEQ ID NO: 265, 267, 269, 271, 273, 275, 276, 278,280, 282, 284 or
 286. 56-58. (canceled)
 59. A pharmaceuticalcomposition, comprising the bispecific antibody according to claim 1 anda pharmaceutically acceptable excipient, carrier or diluent.
 60. Amethod for preparing the bispecific antibody according to m claim 1,comprising: (a) obtaining a fusion gene of the bispecific antibody toconstruct an expression vector of the bispecific antibody; (b)transfecting the expression vector into a host cell by a geneticengineering method; (c) culturing the host cell under conditions thatallow the bispecific antibody to be generated; and (d) separating andpurifying the generated bispecific antibody; wherein the expressionvector in step (a) is one or more selected from plasmids, bacteria, andviruses, and preferably the expression vector is a pCDNA3.4 vector;wherein the host cell into which the constructed vector is transfectedby the genetic engineering method in step (b) comprises a prokaryoticcell, a yeast or a mammalian cell, such as a CHO cell, an NS0 cell oranother mammalian cell, preferably a CHO cell; and wherein thebispecific antibody is separated and purified in step (d) by aconventional immunoglobulin purification method comprising protein Aaffinity chromatography and ion exchange, hydrophobic chromatography ormolecular sieve.
 61. (canceled)
 62. A method for enhancing orstimulating an immune response or function, comprising administering toa patient, subject or individual a therapeutically effective amount ofthe bispecific antibody of claim
 1. 63. A method for treating, delayingdevelopment, or reducing/or inhibiting recurrence of a tumor,comprising: giving or administering an effective amount of thebispecific antibody of claim 1 to an individual suffering from cancer,wherein the cancer comprises mesothelioma, squamous cell carcinoma,myeloma, osteosarcoma, glioblastoma, neuroglioma, malignant epithelialtumours, adenocarcinoma, melanoma, sarcoma, acute and chronic leukemia,lymphoma and meningioma, Hodgkin's lymphoma, Sezary syndrome, multiplemyeloma, lung cancer, non-small cell lung cancer, small cell lungcancer, laryngeal cancer, breast cancer, head and neck cancer, bladdercancer, uterine cancer, skin cancer, prostate cancer, cervical cancer,vaginal cancer, gastric cancer, renal cell carcinoma, renal carcinoma,pancreatic cancer, colorectal cancer, endometrial carcinoma, esophagealcarcinoma, hepatobiliary cancer, bone cancer, skin cancer and bloodcancer, and carcinoma of nasal cavity and sinus, nasopharyngealcarcinoma, oral cancer, oropharyngeal cancer, laryngeal cancer,sublaryngeal cancer, salivary cancer, mediastinal cancer, cervicalcancer, small intestine cancer, colon cancer, cancer of rectum and analregions, ureter cancer, urethral cancer, penile cancer, testicularcancer, vulva cancer, cancer of endocrine system, cancer of centralnervous system, and plasmocytoma.